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Scale-up studies for phase transfer mode of reverse micellar extraction are attempted for the separation and primary purification of bromelain (EC 3.4.22.33) from pineapple (Ananas comosus L. Merryl) waste. Characterization of reverse micelles and mass transfer studies for the real system has been attempted for the first time. Scale-up of the extraction process employing commercial grade surfactant cetyltrimethylammonium bromide (CTAB) and solvent isooctane resulted in purification of 2.43 fold with an activity recovery 81.3%. The reverse micellar size estimated using empirical and geometrical models indicated that the reverse micelles are large enough (R m ¼ 7.2-9.6 nm) to host bromelain molecules that are relatively smaller in size ($1.67 nm). The studies on the kinetics of mass transfer indicated a relatively slower rate (by $34%) of mass transfer in case of back extraction compared to forward extraction. Process scale-up did not significantly affect the extraction efficiency whereas purity of phase components played a major role. The mass transfer across the phases was high in the initial period of mixing for both forward and back extractions.