This study aimed to obtain the condition of maximum bromalein activity in different parts of pineapple plants produced in vitro, by micropropagation. The sStems and leaves of Pérola and Imperial cultivar plants were evaluated after three and eight months of in vitro cultivation in Murashige and Skoog medium without growth phytoregulator, macerated in potassium phosphate buffer at different pH values (5.7, 6.7 and 7.7). Total protein and proteolytic activity were determined in the samples after three- and eight-month cultivation periods. For both the cultivars, the best results were obtained at pH 5.7 in extraction media. Pérola cultivar, showed higher bromelain activity in the leaves cultivated in vitro for three months (0.0194U/mL) while in the Imperial cultivar, it was higher in the stem after eight months (0.0179 U/mL). Imperial cultivar showed higher bromelain activity than the Pérola's.