Dalbergia sissoo is an important timber tree facing mysterious die back and wilting problem. In case of die back, Dalbergia is facing the threat of destruction in its natural habitats due to lack of potential pathogenicity test which is the major bottleneck in pathogen assessment and tree improvement programmes. Isolation of protoplasts was attempted to produce an effective source for the pathogenicity test. This study described a procedure for the rapid isolation, in high yield, of photosynthetically active mesophyll protoplasts from young leaves of D. sissoo. The present study reports the isolation of protoplasts from leaf mesophyll of D. sissoo. Leaf strips were suspended in the enzyme solution for the isolation of protoplast. Different concentrations of enzymes were used to optimize the suitable combination for the protoplast isolation. Enzyme solution turned green after a gentle swirling motion, which indicates the release of protoplasts. Release of protoplast was checked in the solution under the microscope. A combination of filtration, centrifugation and washing was used to purify the protoplasts. The optimum combination of enzymes for protoplast isolation was 1.5% cellulase R-10+ 0.5 % pectinase R-1 after incubation for 6 hr at 28o C. The isolated protoplasts were round and filled with chloroplasts. The size of protoplasts was 20~35 μm. The protoplast yield was 2 × 105 per g of leaf tissue. The protoplast viability as assessed by 0.01% Phenosaphranine staining was 77%. @JASEMKey words: Dalbergia sissoo, Leaf mesophyll, Protoplasts, Phenosaphranine.