Live imaging of animals infected with pathogenic microbes poses a contamination risk to equipment, personnel and other animals. A Caliper animal isolation chamber designed for the IVIS(®) Spectrum imaging system was tested as a containment device for mice infected with microbes assigned to animal biosafety level-3 (ABSL-3). A testing protocol was developed by adapting two published standards to test other equipment in high containment environments. The protocol included quantitative leak-testing of the high efficiency particulate air (HEPA) filters, soap bubble testing of the animal isolation chamber, and pressure decay testing of the complete containment system. HEPA filters were > 99.999% efficient (< 0.001% leakage). When attached to the Spectrum at the normal flow rate of oxygen/anesthetic mix (0.25 L/min), the chamber was positively pressurized at 0.11 inches of water (in H(2)O). No leaks were detected by soap bubble testing at flow rates of 0.25 L/min to 2.0 L/min, generating pressures up to 2.90 in H(2)O. (26-fold increase over normal operating pressure). The complete containment system passed pressure decay testing at 2.0 in H(2)O by sustaining 95% of the initial pressure over a 30 minute period.The Caliper animal isolation chamber provides appropriate isolation for the IVIS(®) Spectrum imaging system. When used as a containment device, it must undergo periodic performance testing, as described here, since it operates under positive pressure. The chamber is an appropriate component of ABSL-3 containment when combined with proper administrative controls and work practices. The testing protocol described here can be used to validate containment devices for other imaging systems or animal species.