The present study was conducted to identify the effects of hormonal treatment on testis development and sperm quality of cultured males of short-finned eel (Anguilla bicolor). A total of 9 male eels were selected (range from 150 and 250 g) with triplicate of each hormone treatments, namely human chorionic gonadotrophin (HCG), pituitary gland (PG), and Ovaprim (OVP). Experiment was conducted for 100 days. Each male was subjected to a total of 20 times hormone injections with five days interval. The initial and final total length and body weight were recorded. Each fish was anesthetized prior to sperm collection. Milt was diluted in seminal fluid plasma for sperm quality assessment. The findings revealed there was no significant difference observed in the final average total length for the HCG, PG, and OVP treatments, with the highest recorded at 48.5±1.6 cm as seen in OVP treatments respectively. In contrast to PG and OVP treatment, the final body weight of male eels treated with HCG was significantly lower (HCG: 185.3±19.4 g, PG: 255.0±43.1 g, and OVP: 260.0±12.6 g). Male eels from PG and OVP were unresponsive to the hormone treatments, but those from the HCG treatment did exhibit a response and a total of 0.8 ml milt with a concentration of 3.12 ± 0.9 x 109 spz ml-1 and the motility percent was 78.23 ± 5.11 % were recoded. Male eels receiving HCG treatment had well-developed testis, while PG treatment still had growing testis as seen by histology analysis; however, those from OVP treatment did not develop as evidenced by the gonad morphology following surgical incision. This study showed HCG able to induce sperm production and enhance testis maturation which is crucial for artificial production and reproductive investigations.