The blacklip rock oyster, Saccostrea spathulata, has significant potential to support oyster aquaculture development in the tropical Asia-Pacific region; however, hatchery production is currently constrained by the absence of a reliable broodstock conditioning protocol. This study aimed to investigate the effects of salinity and diet on blacklip rock oyster S. spathulata gonad development, as well as the subsequent ability to spawn, fecundity, and oocyte quality. Broodstock were conditioned for a period of 8–12 wk and sampled at regular intervals for histological analysis. Results showed that salinity has a significant effect on gonad development, with the greatest proportion of ripe broodstock produced at salinity 25. Gonad development was also dependent on diet. Broodstock fed a diet comprising 100% live microalgae (35% Chaetoceros muelleri; 5% Skeletonema costatum; 20% Tisochrysis lutea, 20% Pavlova spp.; 20% Tetraselmis chui) achieved greater condition indices than those fed a formulated diet and were brought into spawning condition within 5 wk. Further, female broodstock fed the live microalgae diet produced more, and larger eggs compared with those fed a full or partial formulated diet. Based on these results it is recommended to condition blacklip rock oysters at salinity 25 for a period of 5 wk, with a diet comprising live microalgae at a rate of 2–3.5 × 109 Tisochrysis lutea cells–1 oyster–1 day–1. This study provides the first method to condition blacklip rock oyster broodstock and removes a current bottleneck to hatchery production.