Murray et al., in 1993 first demonstrated the ability of HIV-1 PR fused within Gal4 domains to autocatalytically remove itself, leaving behind the two non-functional domains of Gal4 [22]. However, in the presence of PI, or when the PR/Gal4 fusion protein is mutated at the catalytic site, the fusion protein remains intact, retaining its ability to bind to UAS and subsequently activate transcription. This concept has also been applied for the characterization of the autocatalytic activity, of other proteases like the 3C protease of coxsackievirus B3 [28]. We exploited this property of the Gal4 system to express a reporter gene dependent on PR activity, the expression of the reporter gene being inversely proportional to the autocatalytic activity of PR, thus serving as the basis for our assay.