In vitro full expended healthy leaves and sterilized in vivo leaves of pineapple (Ananas cv. smooth cayenne) plants were taken and prepared under aseptic conditions as different sources of explants. Also, different enzymes mixtures, incubation periods, osmotic pressure factors, shaking periods and speeds were concerned in combination with explants sources during protoplast isolation stage. In addition, sieve size and centerfugation speed were evaluated in combination with explants source during purification stage. Moreover, medium type protoplast density, auxin/cytokinin concentration ratio, and antibiotic were tested in combination with explants source during protoplast culturing. It is found that in vitro and sterilized in vivo explants source succeeded in maximizing protoplast yield. Also, using of enzymes mixture consists of 1.0% cellulase + 0.5% macerozyme was superior in increasing protoplast yield Moreover, using of sucrose at rate of 13.6g /100ml as osmotic pressure factor and incubation for 20 hours then, shaking for 15 min with speed rate 75 rpm succeeded in enhancing the highest protoplast isolation of pineapple. Meanwhile, using of 25 μM pore size mesh sieve and centrifugation at the rate of 1000rpm maximized protoplast purification. Moreover, culturing of protoplast KAO and Michayluk medium supplemented with 3.0 mg/l NAA and 0.2 mg/l BAP as well as the combination of antibiotic (0.4 mg/l Ampicilin + 0.1 g/l gentamycin + 0.1 g/l tetracycline) and using protoplast density at the rate of 2.5 x 104 induced the best protoplast viability and development of pineapple explants.