Reliable detection of unwanted organisms in experimental animal colonies is an essential requirement for biomedical research. Since most rodents are housed in Individually Ventilated Cage (IVC) systems which prevent aerogenic transmission of pathogens, soiled-bedding sentinels are commonly used for routine health monitoring. In this study, we investigated whether testing of Exhaust Air Particle (EAP) samples by real-time PCR (EAP PCR) might be a valid alternative to SBS for the detection of H. hepaticus infections. EAP PCR detected H. hepaticus infections at a minimal prevalence of 1/63 cages within one week. EAP PCR provides a sensitive, simple, and reliable alternative to SBS for H. hepaticus identification in laboratory mouse colonies.