Activity simulation protocols offer useful applications in research and practice; however, the specificity of such protocols to basketball game-play is currently lacking. Consequently, this study aimed to develop a game-specific basketball activity simulation protocol representative of typical playing durations and assess its reliability and discriminant validity. The simulation protocol was modified from an original version (i.e., Basketball Exercise Simulation Test) to incorporate regular breaks indicative of time-outs, free-throws, and substitutions. Twelve competitive male and female adult basketball players competing in the fourth or fifth Spanish basketball division underwent repeated trials of the simulation protocol (min. 4 to max. 14 days apart) for reliability analyses. In turn, 13 competitive male (fifth division), 9 competitive female (fourth division), and 13 recreational male adult basketball players completed the simulation protocol to assess discriminant validity via comparisons between sexes (competitive players) and playing levels (males). A range of physical, technical, and perceptual-physiological variables were collected during and following the simulation protocol. Several physical and heart rate variables displayed the strongest reliability (intraclass correlation coefficient [ICC] = 0.72–0.96; coefficient of variation [CV] = 1.78–6.75%), with physical decrement, technical, blood lactate concentration, and rating of perceived exertion (RPE) variables having the weakest (ICC = 0.52–0.75; CV = 10.34–30.85%). Regarding discriminant analyses between sexes, males demonstrated significantly greater physical outputs in several variables and lower RPE compared to females (p < 0.05, moderate-to-large effects). Comparisons between playing levels revealed competitive males had significantly greater physical outputs across many variables, alongside higher mean heart rate and lower RPE than recreational males (p < 0.05, moderate-to-large effects). This study presents a novel game-specific basketball activity simulation protocol replicating actual playing durations and game configurations that might be successfully applied for both training and research purposes. Reliability statistics are provided for several variables to inform end-users on potential measurement error when implementing the simulation protocol. Discriminant validity of the simulation protocol was supported for several variables, suggesting it may hold practical utility in benchmarking or selecting players. Future research on this topic is encouraged examining wider samples of male and female basketball players at different levels as well as additional forms of validity for the protocol.