The majority of assays developed so far, in order to screen for PIs, have been designed in vitro [17–19] in bacterial [20,21], or yeast cells [22]. The lack of assays for PR activity in mammalian cells is, in general, due to the cytotoxic effects of PR [23]. The recently developed mammalian cell-based assays [24,25], although invaluable, may not provide accurate evaluation of the inhibitory effects of putative novel drugs on PR as they are not performed in the natural environment of HIV infection, T-cells. Moreover, most of the assays are not adaptable for efficient and reliable high throughput screening. The growing need for effective assays, which can aid in drug discovery, prompted us to develop a simple yet rapid and straightforward method for monitoring PR activity. The assay, developed in T-cells, will thus facilitate the search for novel PIs/competitors in a cell type naturally infected by HIV.