Yes, you can search for flights on a route through the API to see what airlines and schedules come back, and this helps you decide whether it will work for your business. Most flight APIs let you run test searches without having to commit to anything or start paying fees, so you can check availability on the routes your customers actually want to book. This is smart to do before you build your portal or tell your customers what you offer, because you want to know whether the airlines they expect to fly with will show up in your results.
Keep in mind that the airlines available can shift over time. New airlines might start flying a route, or existing ones might stop, and seasonal routes come and go. The API will show you what is available right now, but you should plan to check back regularly, especially if a customer later asks for a flight you saw during your test searches but cannot find anymore. Some airlines also have blackout dates or limited schedules, so what you see on one search might not be the same on another day.
The best approach is to test the API on all the routes you plan to sell, or at least the most popular ones, and make a note of which airlines show up and how often flights run. If you find that important airlines are missing or flights are too thin on the ground for your market, you will know before you invest time and money in building your portal. Talk to your provider about which suppliers are connected to the API and whether they can add more if you need them.