This study described the subcapsular technique for primary closure castration in donkeys with special regard to its efciency and welfare impacts. The study was conducted on twelve adult male donkeys, allocated randomly into two groups; subcapsular castration (SC) and open castration (OC) groups, whether the donkeys were subjected to surgical castration either by subcapsular or open castration techniques, respectively. Testosterone, cortisol, lactate, glucose, total cholesterol (TC), high density lipoprotein cholesterol (HDL-C), triglyceride (TG), and nitric oxide (NO) were measured before and after castration. Pain-associated behavioral activities were recorded post-castration. The SC was successfully performed in donkeys through a single paramedian scrotal incision. The SC was efcient as OC in reducing testosterone levels. The pain score decreased in the SC compared to the OC over time. The SC was an efcient and reliable technique for primary closure castration in donkeys with minimal postoperative complications and care and good cosmetic, physiological, and behavioral outcomes. It can be an alternative to other castration techniques in equines.