Sperm sexing aims to separate sperm populations in carriers of the “X” or “Y” chromosome. Currently, flow cytometry is a technique that allows greater accuracy; however, it causes structural changes in sperm, reduces viability, and has a high cost. As a result, other methods have been researched, including immunosexing, which uses monoclonal antibodies to detect sex-specific surface antigens. Thus, the objective of this study was to evaluate the immunosexing technique using a monoclonal antibody against sex-specific protein (HY) in the conservation of ram and goat semen in ACP101/102c. Ejaculates from five rams and five goats were collected with the aid of an artificial vagina; they were evaluated and submitted to the immunosexing protocol, according to the manufacturer’s recommendations, using the Monoclonal Antibody Kit specific for mammalian sperm with “Y” chromosomes (HY; HY Biotechnology, Rio de Janeiro, RJ, Brazil). After sexing, the supernatant was resuspended in the cryopreservation diluent: ACP ram (ACP101/102c + 20% egg yolk + 7% glycerol) and ACP goat (ACP101/102c + 2.5% egg yolk + 7% glycerol), packaged in 0.25 mL straws, refrigerated at 4°C, stabilized for 30 min, frozen in liquid nitrogen vapor (-60°C) for 15 min, immersed in liquid nitrogen, and stored in cryogenic cylinders. The samples were evaluated in natura (T1), after immunosexing (T2) and after thawing (T3) for sperm motility subjectively using conventional microscopy (40x). Plasma membrane integrity (IMP) and sperm cell morphology were evaluated by the smear staining technique using eosin-nigrosine dye, and the percentages of healthy and morphologically defect spermatozoa were determined. In the evaluation of ram semen regarding sperm motility and IMP, no statistically significant differences were observed between treatments after sexing in the evaluation of absolute data (P > 0.05), with the difference being observed only between T1 and T2, and T3 (P < 0.05). Regarding the relative percentage and sperm morphology, no statistically significant differences were observed (P > 0.05). Regarding the evaluation of goat semen samples, the motility parameters were consistent with the technique submitted; however, the IMP data did not appear as expected, requiring further evaluation for a better assessment of the technique for this species. The data obtained from ram semen submitted to the immunosexing protocol, regarding the absolute evaluation of motility and IMP, demonstrated that the non-sexed semen (T1) was superior to the sexed treatments (T2 and T3); however, it is noteworthy that freezing started with approximately 50% of the cells, since the immunosexing technique results in a loss of viability of approximately 50% of the sperm, which corresponds to the ratio of sperm carrying the X chromosome. In addition, when the data in this study were transformed into relative values, no statistical differences were observed, indicating that the immunosexing protocol, as well as the freezing protocol, did not significantly affect the quality of ram sperm cells. In relation to the immunosexing of goat semen, future studies should be conducted in vitro to define a more appropriate protocol for the species and, in addition, in vivo studies should be performed to prove the quality of the technique. It was concluded that the immunosexing process using a monoclonal antibody against sex-specific protein (HY) associated with the use of powdered coconut water diluent (ACP101/102c) in the cryopreservation of semen proved to be efficient in the in vitro evaluation of ovine species.(AU)