Reliable detection of unwanted microbial agents is essential for meaningful health monitoring in laboratory animal facilities. Most rodents at our institution are housed in IVC rack systems to minimize aerogenic transmission of infectious agents between cages. The most commonly used rodent health monitoring systems expose live sentinel rodents to soiled bedding collected from other rodent cages on IVC racks and subsequently test these soiled-bedding sentinels for evidence of infection with excluded agents. However, infectious agents might go undetected when using this health surveillance method, due to inefficient organism shedding or transmission failure. In 2016, our institution switched the health monitoring methodology for the majority of our SPF rodent colonies to real-time PCR testing of environmental samples collected from the exhaust plenums of IVC racks. Here we describe our rationale for this conversion, describe some interesting health monitoring cases that arose soon after the conversion, and discuss a potential problem with the conversion—residual nucleic acids. We compared cost and implementation effort associated with 2 sampling methods, sticky swabs and in-line collection media. We also compared the ability of these 2 sampling methods to detect 2 prevalent microbes in our facilities, Helicobacter and murine norovirus. Our institution-wide switch to health monitoring by real-time PCR assay of exhaust air dust samples thus far has provided a sensitive, simple, and reliable approach for maintenance of SPF conditions in laboratory rodents and has dramatically reduced the use of live sentinel animals.