The isolation of protoplasts from diverse plant species is a widely employed technique. The purpose of this work is to develop an efficient system for isolating and purifying mesophyll protoplasts from Daucus carota. Main factors influencing the qualitative and quantitative dimensions of protoplast isolation procedures attempted to be optimized, using the well-established protoplast fusion technique as the foundation for the comprehensive analysis, including sorbitol concentration during the preplasmolysis stage and the duration of the enzymolysis process, those key variables affect the yield and survivability of the protoplasts. This research employed "Vil-1" carrot leaves as the primary source material to isolate protoplasts through enzymolysis. The data revealed that higher concentrations of sorbitol led to increased protoplast yield, with the optimal concentration being 0.5 M, which resulted in up to 95% protoplast vitality. Furthermore, prolonging the enzymolysis duration to 6 hours maximized both protoplast yield and vitality. The optimal conditions for isolating protoplasts were determined to be 0.5 M sorbitol pre-treatment for one hour, combined with a mixture of 1% cellulase, 0.1% pectinase, and a 6-hour incubation period.