Background: AIDS in humans, which is caused by the HIV remains among the leading causes of death worldwide. Objective: The study was undertaken to investigate the HIV-RT inhibitory activity of Phytotesrols extracted from roots of Aerva lanata. Methodology: Sequential maceration method was performed for preparation of extracts using hexane, chloroform, ethyl acetate, acetone and methanol solvents. RetroSys HIV-1 RT (Innovagen, Sweden) kit was used to determine the HIV-RT inhibitory activity of all solvents extracts. Results & Discussion: All extracts showed significant inhibitory activity. Chloroform extracts shows highest inhibition of recombinant HIV-RT (89.0%) at 2 and mg/ml concentration. Phytosterols isolated from chloroform extracts. These results were remarkable and can be used to develop a new drug for HIV treatment or other infectious diseases caused by the pathogenic micro-organisms, for which plant sterols may play a distinctive role. Conclusion: Further work is in progress to evaluate the advanced spectroscopic studies are required for the structural elucidation, identification and characterization of the active compound from this plant.