Sperm capacitation is a critical process for fertilization. This process involves several biochemical and physiological changes that have been extensively studied, but little is known about the changes that occur in the sperm’s proteomic profile. This work aims to analyze the effect in vitro capacitation had on the proteome and mitochondrial parameters of bull spermatozoa. Samples of non-capacitated (NC) and in-vitro capacitated (ICV) sperm were analyzed using flow cytometry to assess sperm viability, mitochondrial membrane potential (MMP), and reactive oxygen species (mROS). Proteome was evaluated using SWATH-MS. In-vitro capacitation induced a significant decrease in sperm viability, high MMP, and a significant increase in mROS production. Within the group of living spermatozoa, the capacitation process induced a significant decrease in healthy mitochondrial spermatozoa, as well as a significant increase in mROS production, without affecting the MMP intensity