The isolation and purification of Tat protein from bacterial lysate using avidinbiotin interaction in microfiltration membranes have been reported in the literature. To increase the efficacy of the technique, improvements in flux, Tat separation efficiency, and processing time are essential. In the current research work a pre-filtration step was introduced to remove unwanted high molecular weight proteins and other impurities from feed prior to affinity membrane separation. Significant enhancement in flux and separation efficiency of Tat was observed. Processing time was also reduced significantly. For example, with UF pretreatment step the total Tat recovery was around four times higher (with processing time 25% lower) than that observed with the untreated feed. The quality of purified Tat was analyzed by SDS-PAGE, Western Blot, and biotin analysis. Flux behavior in affinity separation was described by model equations.