This study aimed to identify potential protein biomarkers for antemortem diagnosis of rabies in dogs, which are the principal reservoir hosts of the rabies virus. Lyssavirus rabies, the prototype species, effectively evades the host immune response allowing the infection to progress unnoticed until the onset of clinical signs. At this stage, the disease is irreversible and invariably fatal, with definitive diagnosis possible only post-mortem. Two hundred and thirty-one samples of brain tissue, cerebrospinal fluid, and serum were collected from apparently healthy dogs brought for slaughter for human consumption in South-East and North-Central Nigeria. All the brain tissue were subjected to a direct fluorescent antibody test to confirm the presence of lyssavirus antigen, and 8.7% (n = 20) were positive. Protein extraction and quantification was performed on 20 rabies-infected and -uninfected samples from each sample type, and only 10 rabies-infected and -uninfected samples were selected from each sample type as they had sufficient protein quantity for further sample preparation. Reduction and alkylation of the selected samples was performed and on-bead HILIC sample clean-up and tryptic digestion followed thereafter. The resulting peptides from each sample were injected into the EvoSep One LC system, coupled to the timsTOF HT mass spectrometer, using the standard dia-PASEF short gradient data acquisition method. Data was processed using Spectronaut (v19) and an unpaired t-test was performed to compare identified protein groups (proteins and their isoforms) between the rabies-infected and uninfected brain tissue, cerebrospinal fluid, and serum samples.