Updated project metadata. Bats are increasingly studied as model systems for longevity and as natural hosts to some virulent viruses. Yet our ability to characterize immune mechanisms of viral tolerance and to quantify infection dynamics in wild bats is often limited by small sample volumes and few species-specific reagents. To address this, we demonstrate how proteomics can overcome these limitations by using data-independent acquisition-based shotgun proteomics (i.e., bottom-up proteomics) to survey the serum proteome of 17 vampire bats (Desmodus rotundus) from Belize. We focused this work on vampire bats, a species that has an obligate diet of blood and feeds on prey as diverse as sea lions, tapirs, livestock, and even humans, providing numerous opportunities for transmission of viruses (e.g., rabies virus, adenovirus, herpesvirus) to and from these recipient hosts. Using just 2 μL of sample and relatively short separations of undepleted serum digests, we identified 361 proteins across five orders of magnitude. We also used known bat virus proteomes to identify Rh186 from Macacine herpesvirus 3 and ORF1a from Middle East respiratory syndrome-related coronavirus, indicating that mass spectrometry-based techniques show promise for pathogen detection. Our results demonstrate the feasibility and capabilities of serum proteomic analyses in wild bats, including possibilities to simultaneously detect host immunological components and viral infection as well as to establish preliminary ranges of vampire bat proteins for comparison with other mammalian blood proteomes. Overall, these results can be used to design targeted mass-spectrometry assays to quantify immunological markers and detect pathogens. More broadly, our findings also highlight the application of proteomics in advancing wildlife immunology and pathogen surveillance.