Sperm capacitation is essential for fertilization and involves coordinated changes in membrane organization, ion fluxes, and intracellular signaling. However, commonly used detection methods may reflect different biological events, which can be strongly influenced by experimental methodology. This study critically evaluated fluorescence-based approaches for assessing capacitation in boar spermatozoa, focusing on their specificity, interpretative limits, and methodological sensitivity. Ejaculated boar spermatozoa were incubated under in vitro capacitating conditions in TALP medium. Selected samples were subsequently treated with calcium ionophore to induce the acrosome reaction (AR). Phosphotyrosine (PTyr) immunofluorescence was assessed using five fixation and labeling protocols, acrosin redistribution was evaluated with the ACR.2 antibody, calcium ion redistribution was assessed using chlortetracycline (CTC) fluorescence, and acrosomal responsiveness was monitored by peanut agglutinin (PNA) lectin labeling. PTyr immunofluorescence was highly dependent on fixation protocol, indicating marked methodological sensitivity. Acrosin immunodetection revealed a clear capacitation-associated redistribution from weak or diffuse staining to a well-defined acrosomal pattern, whereas ionophore treatment caused a pronounced signal loss consistent with acrosomal exocytosis. PNA labeling confirmed that capacitation alone did not increase spontaneous acrosome loss, whereas ionophore treatment induced a robust AR. CTC staining showed a significant shift from whole-head pattern to acrosome in TALP-treated spermatozoa, indicating capacitation-associated Ca²⁺ redistribution. Together with CTC and Western blot data, these findings show that sperm capacitation status should be evaluated using multiple complementary markers rather than a single gold-standard assay.