GAE: is putting all handlers in main.py gonna make my app slow?
No, that doesn't affect the speed. Your code needs to be loaded anyway, so it makes no difference if it's all in one file or not. It will of course make the file more complex, but that's your problem, not GAE's.
I'm building a web application using GAE.
I've been doing some research by my own on GAE python project structures,
and found out that there isn't a set trend on how to place my handlers within the project.
As of now, I'm putting all the handlers(controllers) in main.py, 
and make all urls (/.*) be directed to main.application.
Is this going to make my application slower?
Thank You!