In order to capitalize on the potential of using protoplast fusion technology to solve breeding difficulties within the framework of breeding programs for the creation of F1 carrot hybrids, factors impacting the qualitative and quantitative aspects of the protoplast isolation method were investigated. This work uses "Vilmarin" carrot leaves to isolate protoplasts through enzyme hydrolysis. Two main effectors—the concentration of sorbitol in preplasmolysis and enzymolysis time, affect the production and vitality of protoplast. The hemacytometer and FDA staining were used to determine carrot protoplast production and vitality, respectively.