The carmine spider mite Tetranychus cinnabarinus (Boisduvals) is a serious pest on vegetable and fibre crops often causes considerable loss in yield. Previous reports have shown that the increase in activity of glutathion-S-aromatic groups transferases (GSAT) is an important mechanism of pest resistance to organophosphorus pesticides. GSAT and GSH-S-alkyltransterases of GSH-S-transferases in the pest body are mainly associated with pesticide resistance. In this paper, the regression method was adopted to select the optimal combination of different buffer pH value, reaction temperature, reaction time and substrate concentration for determining the enzyme activity in spider mites. Five levels of the four factors were tested and the experiments were conducted under the condition of interaction between the factors. The results showed that GSAT had the highest activity under the following combination of the four factors: buffer pH 7.8, reaction temperature 25°C, reaction time 35 minutes, and concentration of substrate 0.125 M. This combination is therefore considered as optimal conditions for determining the activity of GSAT of the spider mite, and can be used to determine the change of enzyme activity caused by pesticide resistance in these mites.