Timely and accurate detection of murine pathogens is essential in contemporary biomedical research. Cost, accuracy, and reproducibility of test results are frequent concerns when initiating an on-site serology program. This study was conducted to evaluate the advantages of on-site serology performed by enzyme-linked immunosorbent assay (ELISA) versus pathogen surveillance conducted off-site by a commercial vendor. We divided 92 sentinel mouse serum samples and tested them in parallel for a panel of 10 murine pathogens at our institution and by an off-site vendor. On-site testing was performed with commercially available test kits and according to the kit manufacturer's directions, whereas serum samples for off-site testing were prepared according to the vendor's specifications. Results from the 2 testing strategies were compared, and a good beyond- chance level of agreement was demonstrated by means of the kappa test (kappa = 0.86). The turn-around time between sample preparation and results availability for on-site ELISA was 16 h versus 72 h for off-site testing. On-site ELISA demonstrated considerable cost reduction, ranging from 15.10% to 43.33% depending on the number of agents being tested. This study demonstrates the accuracy and time- and cost-effectiveness of on-site ELISA as well as its potentially valuable role in achieving more timely and efficient disease surveillance and control programs in contemporary biomedical research facilities.