Reverse micellar extraction (RME), a liquid-liquid based separation is a versatile tool for protein purification. A statistical approach was employed for the purification of recombinant glutaminase free anti-cancerous enzyme viz., L-asparaginase II to evaluate the effects of RME in current study. The cationic system (CTAB/iso-octane/hexanol/butanol) was used in RME to optimize both forward and backward protein extraction efficiency. By adapting Taguchi's orthogonal array (OA), maximum forward extraction efficiency (FEE) of 86.98% with 84.82% enzyme activity recovery and 1.04 times purification fold achieved with the optimized parameters. Under the optimal levels, the back extraction efficiency (BEE) was observed to be 96.97% with 93.07% enzyme activity recovery and 1.38 times purification fold. Further, mass transfer kinetic studies of RME indicated the mass transfer coefficients of forward and backward extraction to be 0.049 min(-1) and 0.036 min(-1) respectively.