Application issues are the trickiest to sort out, especially if it’s a multi-tiered application, put load balancers, QOS, and rules based routing and you have a recipe for a migraine.
In this video walk you through a web-based application issue. I would suggest you not worry about the application specifics, but more on the methodology.
For example, I start from the clients’ end of the network to better understand the issue. Secondly, I test to the server via different tests.
I also ask that you occasionally ‘calibrate’ your network tools. What I mean is to perform a packet capture to validate what the network tool is reporting.