This study investigates the impact of three precipitation methods, ammonium sulfate (40%), acetone, and acetone with trichloroacetic acid (TCA) on the yield and activity of protease extracted from Averrhoa carambola (starfruit) at various maturity stages (unripe, semi-ripe, and ripe). The study employed response surface methodology (RSM) to optimize hydrolysis conditions, specifically temperature, and pH, for maximum proteolytic activity. The proteases were incubated in buffers at pH values ranging from 3.5 to 8.5 for 12 hours, and the proteolytic activity was assessed. Additionally, temperature effects were evaluated by incubating proteases at temperatures between 40◦C and 90◦C. The storage stability of the proteases was monitored over a 7-day period at temperatures <4◦C. The results revealed that ammonium sulfate precipitation (40%) yielded the highest proteolytic activity (PA), particularly at the unripe stage, where it reached 0.34 units. Conversely, acetone with TCA precipitation exhibited the highest specific activity, with values of up to 2.38 U/mg at the ripe stage, indicating that although the total protease yield was lower, the enzymes extracted were more efficient per unit of protein. Acetone precipitation showed intermediate values for both PA and specific activity. Numerical optimization identified the optimal conditions for proteolytic activity as 65◦C and pH 6.5, achieving a maximum activity of 0.862 units/mL. Despite the promising proteolytic activity under optimized conditions, a significant reduction in enzyme activity was observed during a 7-day storage period at <4◦C. This finding underscores the importance of considering both total proteolytic activity and specific activity when selecting the optimal protease extraction method. Overall, this study demonstrates that ammonium sulfate precipitation is the most effective method for extracting protease from starfruit, particularly at the unripe stage for higher yield, while acetone with TCA precipitation offers advantages in producing more active proteases, suitable for applications requiring enzymatic efficiency.