The utility of dried blood spot (DBS) sampling technology for rodent colony health monitoring was investigated using a two-tiered approach. In the first approach, groups of mice and rats were experimentally infected. Mice were inoculated with epizootic diarrhea of infant mice virus (EDIM), mouse hepatitis virus (MHV), murine norovirus (MNV), mouse parvovirus (MPV), or Theiler's murine encephalomyelitis virus (TMEV). Rats were inoculated with Kilham rat virus (KRV) or rat theilovirus (RTV). Paired DBS and serum samples were collected at 2, 4, 6, and 12 weeks post-inoculation. In the second approach, designed to evaluate real-world conditions, over 1000 mouse and rat paired DBS and serum samples were collected from collaborating North American institutions. Dried blood spot and serum samples were evaluated by multiplex fluorescent immunoassay (MFI) against a comprehensive panel of infectious disease assays and data were compared for magnitude of MFI signal, signal to noise ratio, correlation, and stability. Results from these studies demonstrated that the magnitude of the mean MFI signal from the DBS samples was equal to or higher than the corresponding serum sample. Moreover, the assay signal-to-noise ratio from the DBS samples was equal to or higher than the corresponding serum samples. Evaluation of a large cohort of paired clinical DBS and serum samples showed 100% diagnostic correlation and improved specificity. Finally, stability studies showed no degradation of MFI signal when DBS samples were protected from humidity and stored at room temperature or 4°C for up to 14 days or when stored at -20°C for up to 3 months, compared to controls. A moderate decrease in MFI signal was encountered when samples were stored at 37°C in 95% humidity for 24 hours; however, all samples remained diagnostically positive.