This study was conducted to monitor the production of protease enzyme derived from specific parts of medicinal plants found in Siruvani Hills, Tamil Nadu, Western Ghats. The main objective was to optimize the kinetic parameters of the enzyme in order to establish a large-scale production process. Among the six isolates, Aspergillus flavus and Aspergillus niger were identified as potential candidates for enzyme production using the plate assay screening method. The results revealed the highest enzyme production by these isolates at different temperatures and pH levels. Notably, Aspergillus flavus exhibited the highest activity in the optimized medium at pH 8.0 and a temperature of 30 ºC, while Aspergillus niger showed the highest activity at pH 7.0 and a temperature of 35 °C.