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<i>Streptococcus uberis</i> is a common bovine mastitis pathogen in dairy cattle. The rapid identification and characterization of antimicrobial resistance (AMR) in <i>S. uberis</i> plays an important role in its diagnosis, treatment, and prevention. In this study, matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) was used to identify <i>S. uberis</i> and screen for potential AMR biomarkers. <i>Streptococcus uberis</i> strains (<i>n</i> = 220) associated with bovine mastitis in northern Thailand were identified using the conventional microbiological methods and compared with the results obtained from MALDI-TOF MS. <i>Streptococcus uberis</i> isolates were also examined for antimicrobial susceptibility using a microdilution method. Principal component analysis (PCA) and the Mann–Whitney U test were used to analyze the MALDI-TOF mass spectrum of <i>S. uberis</i> and determine the difference between antimicrobial-resistant and -susceptible strains. Using MALDI-TOF MS, 73.18% (161/220) of the sampled isolates were identified as <i>S. uberis</i>, which conformed to the identifications obtained using conventional microbiological methods and PCR. Using PCR, antimicrobial-resistant strains could not be distinguished from antimicrobial-susceptible strains for all three antimicrobial agents, i.e., tetracycline, ceftiofur, and erythromycin. The detection of spectral peaks at 7531.20 <i>m/z</i> and 6804.74 <i>m/z</i> was statistically different between tetracycline- and erythromycin-resistant and susceptible strains, respectively. This study demonstrates a proteomic approach for the diagnosis of bovine mastitis and potentially for the surveillance of AMR among bovine mastitis pathogens.