Adenovirus-based products are being developed for human use as vaccine constructs and gene therapy vehicles. In order to operate by cGMP guidelines and to address biosafety concerns, it is imperative to ensure that adequate decontamination procedures are implemented in adenovirus manufacturing facilities. Liquid decontamination procedures based on heat treatment are relatively easy to employ in a manufacturing setting. In this study, we have conducted small-scale experiments to determine the effectiveness of thermal inactivation procedures on adenovirus type 5. We have determined the robustness of the assays used to measure sample potency with regard to different sample matrices and adenovirus constructs. Greater than eight logs of reduction in adenovirus type 5 potency may be obtained upon exposure of the sample to temperatures greater than 70 degrees C and times longer than 20 min.