AbstractFlow cytometry is a powerful tool for the analysis of cell samples formed of multipopulations, such as spermatozoa. In recent years, multiparametric cytometers have evolved, allowing the study of different cellular characteristics, such as protein expression, DNA analysis, or mitochondrial activity. Whether using traditional fluorescent dyes or fluorophore‐conjugated antibodies, each cell or cellular component is individually stained, the sample is analyzed at high velocities, and then is displayed and interpreted in a dot‐plot. We hereby describe the procedure to perform a multiparametric flow cytometry analysis in equine spermatozoa using three sources of excitation and polychromatic flow cytometry for the detection of 4HNE, a lipid peroxidation adduct (by anti‐4HNE antibody), apoptotic markers (by caspases 3 and 7 activity), and live/dead spermatozoa (by ethidium‐homodimer) excluding the debris with Hoechst 33342 staining and gating. This multiparametric analysis allows the simultaneous detection of different spermatic parameters, providing useful information for the characterization of a seminal sample and fertility estimation. © 2023 Wiley Periodicals LLC.Basic Protocol: Determination of viability, caspase 3 and 7 activity, and 4‐hydroxynonenal in equine spermatozoa by flow cytometry