Hypoosmotic swelling test (HOST) is used for evaluating the plasma membrane function and fertilizing ability in mammal spermatozoa. However, HOS solutions and experimental conditions have not been determined clearly for assessing canine spermatozoa. This study was conducted to examine the HOS solutions and assay conditions, including incubation time (30 to 120 min), storage temperature (4, 17 and $20^{circ}C$), semen status (fresh and frozen). Maximum spermatozoal plasma membrane swelling was obtained in an 150 mOsm Na-citrate/Fructose solutions with an incubation time for 45 min. The storage temperature and semen status affected the percentage of HOS positive spermatozoa. The HOS test adapted to canine spermatozoa in this study was simple and highly consistent assay with good repeatability. The optimal condition of HOST in canine spermatozoa is an 150 mOsm Na-citrate/Fructose solutions with an incubation time for 45 min regardless of semen storage temperature and semen status.