A system and method for accurately and efficiently testing a HEPA filter in a sterile environment, such as a clean room, by creating a closed-loop system. The system includes a multi-port interface including at least a challenge port and a verification port, each port being in communication with an area upstream from the HEPA filter. Through the challenge port, test substances are introduced to a supply air duct that is in communication with a HEPA filter. Through the verification port, a concentration of the test substances can be measured for comparison with a concentration that passed through the HEPA filter, i.e. downstream from the HEPA filter. As such, test substances can be dispersed throughout the air conditioning system before interacting with the HEPA filter, and accurately measured. The closed loop between the ports provides an accurate measurement of the concentration of test substances to test the HEPA filter.