Over the last two decades, mass spectrometry (MS)-based proteomics techniques have emerged as powerful tools for enabling complex biological systems to be pieced together. However, the adoption of proteomic approaches into studies of animal physiology lags behind the human biomedical research. Traditionally, veterinary proteomics investigations are often conducted using laborious 2D-gel techniques, with difficulty in resolving proteins within “gel spots”. Recently, SWATH-MS approach has emerged as reliable and high throughput alternative. We have applied SWATH-MS based quantitation workflow on a study assessing the changes in bull semen characteristics after an acute temperature increase by scrotal insulation (SI), electroejaculated Bos indicus Brahman bulls. Brahmans bulls (n=6) were split into two groups of three bulls with and without SI, and semen was collected by electroejaculation at three days intervals from before (-10 d), until 74 d after initiation of SI. A subset of samples identified by peripheral semen characteristics, were selected for SWATH-MS assessment, at 21 and 24d after SI, where there were significant differences in the “semen quality” between the two experimental groups. DDA acquisition identified a total of 418 proteins from a pool of seminal plasma samples, in order to obtain the base spectral library. Subsequent SWATH-MS analysis reproducibly quantified 158 proteins and a significant difference in magnitude of change in 29 proteins (adjusted P<0.05) was found between the two treatment groups. SWATH-MS result was compared to previously acquired 2D-gel data on the same set of samples revealing vast agreement between these two techniques, although SWATH-MS showed a greater resolution of changes observed in the proteome. The change in seminal plasma proteome profile, stimulated by an acute heat insult, has direct ramifications on maturing spermatocytes during spermatocytogenesis, subsequently semen quality. The verification of these biomarkers assists management of bull fertility from testicular insult to recovery during acute environmental heat stress.