Over the last two decades, proteomics based mass spectrometry (MS) techniques have emerged as powerful tools and have advanced our understanding of the complex biological systems of living organisms. However, the adoption of proteomic approaches in the clinical veterinary field lags behind than in the human medical field. Traditionally, veterinary proteomics investigations are conducted using 2D gel techniques that is laborious and require extensive optimization to achieve reproducible results. Recently, SWATH-MS approach has emerged as reliable alternative but application into veterinary filed is compromised by the limited protein databases available for many animal species. We have enabled SWATH-MS based quantitation in a recent study assessing the change in sperm characteristics occurring after an acute temperature increase obtained through scrotal insulation (SI), in regularly electroejaculated Bos indicus Brahman bulls. A total of six Brahmans were included and SI was applied to three of the bulls for 48 h, and semen was collected by electroejaculation at three days intervals from before (-10 d), until 74 d after initiation of SI. In particular, 418 proteins were identified from a pool of seminal plasma samples using DDA acquisition in order to obtain the spectral library. Subsequent SWATH-MS analysis reproducibly quantified 158 proteins. Significant difference magnitude of change (adjusted P<0.05) in protein concentration between the two treatment groups were found in 29 out of 158 proteins. SWATH-MS result was compared to previously acquired 2D gel data on the same set of samples revealing vast agreement between these two techniques.