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Castration refers to induced sterility via physical, chemical, or hormonal methods. Chemical castration is an efficient and reliable technique in contrast to other sterilization procedures as it is less painful to physical methods and cost‑effective to hormonal methods. Azadirachta indica (neem), is a charismatic plant as its leaves possess anti-inflammatory, anti‑microbial, and anti-androgenic chattels. To abate the escalating human population in South Asia, neem oil and neem leaf extract have been effectively used as a contraceptive agent. The key determinant of the current study was to evaluate Neem as a chemical sterilizing agent, (either necrotic or apoptotic), in dogs injected intratesticular in comparison to a hypertonic saline solution. Pre- and post-injection testicular width size and blood samples for serum testosterone levels were collected on alternative days. Results disclosed substantial changes in testicular width size, histopathological profile, and serum testosterone level. A non-significant (P > 0.05) pre-injection testicular width readings in contrast to a significant increase (P < 0.05) three days post-injection was noted in all the competitive groups. The mean values recorded for testicular width size at the end of the trial study via neem leaf extract, 30% HSS and, control groups were 27.7362 ± 2.3315mm, 30.9594 ± 4.6861mm, and 24.5023 ± 2.5387mm, respectively. A declining trend, regarding serum testosterone level being statistically significant (P < 0.05) was recorded in treated groups (A, B) in contrast to the control group (C) as the values were 1.5357 ± 0.7819ng, 1.2669 ± 0.9095ng, and 2.4517 ± 0.1827ng in groups A, B, and C, respectively. Histopathological findings advocated the presence of apoptotic bodies in the neem treated group whereas the presence of degenerated interstitial cells, necrosed seminiferous tubules, damaged germinal epithelium, and ceased spermatogenesis was also studied in both competitive groups. Thus, the apoptotic effect and anti-inflammatory property of neem leaf extract resulted in less painful castration and verified Azadirachta indica as a better substitute for chemical castration in contrast to hypertonic saline solution.