Before getting into the details on actual Trainz operations, I always take that 10,000 foot view first. I've written this in many places here, but typical of the search mechanism, it's difficult to find.
Railroads need a purpose to exist. That raison d'etre can be anything whether the railroad serves coal mines and breakers and sends of mine contents to long distant power plants all the way to a full-fledged Class I serving a whole region with a bit of everything including through passenger service, or a tourist line operating on a once forgotten branch. A branch line operation too could connect to a mainline somewhere whether it's a portal, or an actual mainline, long or short, with portals on either end. My Gloucester Terminal Electric, created originally from George Fisher's Gloucester Terminal, is just that as a passenger trolley operation with some freight switching operations. Let the imagination run wild, but pick a theme. I'll cover this in detail.
When developing a route, come up with a theme and stick to this. The actual route-building portion will take forever so I won't go into details about that, but develop a back history for the railroad's existence. This might sound over the top for a virtual railroad or model railroad, but what it does is keep the railroad to a theme so that it's easy to build and you don't have weird things, unless you want them, like a giant coal mine next to skyscrapers next to something like the Grand Canyon. The other thing this does is keep the building consistent so that the coal mine in the mountains has that look and feel and that interchange with the mainline can develop as well as give you a goal to work towards so that the bowl of spaghetti routes don't develop.
Look at maps...
This is the most invaluable thing to do. We're lucky today with so many mapping sources available online. Bing, Google Earth, and other historic map services, not only provide a wealth of information including past and present railroads if they exist, but they also can give us something to look at to plan a fictional route. Using TransDEM, for example, the imported route can then be explored on the ground so to speak, and lines built where none exist. I've done this with a few routes I've started and still working on. A map may have some spurs and a string of power lines going off towards somewhere, for example. Since I'm looking at a topographic map in this case, I noticed that the lines followed a valley and a road, and the route is fairly flat with some hills here and there requiring some extra surveying. With a little bit of manipulation, this power line route becomes a railroad. In this case, I'll keep the roads mostly intact as well as the small towns where I can put in small industries that would be typical for the area and build upon that.
Look at various railroad operations...
https://en.wikipedia.org/wiki/List_of_common_carrier_freight_railroads_in_the_United_States
This is another invaluable source. I've read bits and pieces of it over the years, but this rather comprehensive list contains various railroad histories, some in better detail than others, and other things such as commodities and industries carried, interchange partners, trackage-rights, and other good stuff that can play into a route. This stuff mentioned here doesn't mean your routes have to be prototypical. You can use these bits and pieces to develop a fictional railroad.
Actually write the history of your new route...
This sounds corny, but it actually helps nail down everything that we've discussed here. My old Enfield and Eastern, a route I started in December 2003, has an extensive history as a regional rail line that connected a mill city to an ocean port. The Gloucester Terminal Electric too has some history behind it as well. Here's a bit on the GTE:
This route was once an electric switching railroad built in the early 1900s to serve the various industries and docks located in and around Gloucester. The line dieselized in the 1950s and ran with small switchers due to the aging infrastructure which required a major overhaul. Under various management and owners, service declined and business moved to trucks, however, there was still a need for the railroad. With the ever increasing traffic in and around Gloucester (In real life it's truly horrid), the trolleys returned as modern LRVs imported from Germany, and the freight service was expanded with help from the state and the Cape Ann regional business association.
Freight is handled by diesel road switchers of various vintages and some still retain their former owner's reporting marks (Thanks to Joint Rail)... The commodities handled by the GTE are: Seafood, frozen foods, manufactured goods, oil, brewery-related commodities, propane, and plastic pellets, which are interchanged with the Boston to Rockport mainline. (In reality the Rockport branch terminates at a passenger station, but here there are portals to provide operating interest) where there are through freights of various sorts as well as Boston and Rockport-bound commuter trains.
Anyway...