Semen quality is one of the key factors leading to the success of artificial insemination, however, it is rich in long-chain polyunsaturated fatty acids which are involved in the proliferation of reactive oxygen species and lipid peroxidation in sperm, which causes sperm destruction by lipid peroxidation and can lead to reduced fertility with reduced antioxidant capacity. Some suggested the use of dietary additives to enhance antioxidant capacity to improve sperm quality and fertility in roosters. Therefore, the study was aimed to evaluate the effect of vitamin C (vitC) supplementation in combination with protease on growth performance and sperm quality in Noi crossbred cockerels from 24 to 36 weeks of age. The experiment was conducted with a total of 36 Noi crossbred cockerels with initial bodyweight (BW) 1500 - 2150 ± 167.5g/bird, arranged in a completely randomized design with 4 treatments, respectively. The treatments were Control included a basal diet (KPCS); C75Pro included KPCS + (125mg Protease +75mg vitC)/kg of feed; C125Pro included KPCS + (125mg Protease + 125mg vitC)/kg of feed; and C250Pro included KPCS + (125mg Protease + 250mg vitC)/kg of feed. Each treatment was repeated 9 times. Each repetition was 1 cockerel. The results showed that the BW of 32 and 36 week old cockerels had a statistically significant difference among treatments (P < 0.05), the highest BW at 32 weeks was C125Pro (2278g) and at 36 weeks was C250Pro (2500g) compared to control; Feed conversion coefficient from 24 to 36 weeks of age had a statistically significant difference among treatments (P < 0.05), the most effective in C250Pro (1.46). Regarding the training time, there was no statistically significant difference among the treatments (P > 0.05), but in C250Pro, the training time and the non-response rate were better than the rest of the treatments. Regarding semen pH, sperm activity, survival rate, morphological ratio and concentration, there were statistically significant differences among treatments (P < 0.05), with the best sperm quality in C250Pro compared with other treatments.