In an attempt to achieve optimum conditions for protoplast isolation, purification and fusion in two tomato (Lycopersicon esculentum) hybrids (Hatouf and Barakah). Several experiments were carried out. Callus was induced on leaves explants and maintained on MS medium supplemented with (2.0) mg/l BAP and (0.5) mg/l NAA. Optimum conditions for protoplasts isolation from leaf and callus were investigated. Results showed that the optimum conditions for protoplasts isolation was achieved using enzyme mixture (pectinase 0.5%, cellulase 2.0% and himecellulase 2.0%), at pH 5.4 and temperature 25°C for 24 hrs. and the number of isolated protoplasts from leaves under these conditions were 1950000 protoplast/ml and 3500000 protoplast/ml from callus. Results showed that using several temperatures (20, 23, 28 or 30°C) gave protoplasts number isolated from leaves (600000, 1800000, 2150000 and 450000 protoplast/ml) respectively and (1500000, 3250000, 3750000 and 800000 protoplast/ml) from callus respectively. The use of several pH values (5, 5.2, 5.8 or 6) gave protoplasts number (900000, 1700000, 1550000 and 700000 protoplast/ml) from leaves and (1750000, 3100000, 2600000 and 1100000 protoplast/ml) from callus respectively. While the use of several incubation periods (18, 48 or 72hrs.) gave protoplasts number isolated from leaves (1500000, 1400000 and 600000 protoplast/ml) and (2100000, 2350000 and 950000 protoplast/ml) from callus. The optimum conditions for protoplasts fusion between two tomato varieties (Hatouf and Barakah) were carried out. Results showed that adding deferent molecular weights of Polyethylene glycol (2000, 4000, 6000 or 8000 m.wt) gave fusion percentage (25, 35, 50 and 15%). Results showed that the use of different chemical fusogens (NaNO3 and Ca++ at high pH 10.5) gave fusion percent (10 and 35%) respectively. Optimum conditions for protoplast fusion were achieved by adding Polyethylene glycol (PEG) 6000 (m.wt) + Ca++ at 10.5 pH, since such circumstances recorded the highest percentage of fused protoplast (60%).