Assays and kits for the detection of phenotypic resistance of a retrovirus to reverse transcriptase inhibitor-drugs in a biological sample. The assays are based on the direct analysis of the susceptibility of retroviral reverse transcriptase to inhibition by a reverse transcriptase inhibitor drug. The enzymatic activity of the reverse transcriptase is determined by measuring the DNA product produced when an RNA template and a first complementary DNA primer from a suitable region of the encephalomyocarditis virus genome are incubated with a biological sample containing reverse transcriptase in the presence of the drug to which resistance is being determined. The DNA product is amplified and detection of the amplified DNA indicates resistance to the drug employed in the assay. Detection of relatively greater amounts of amplified DNA when certain drugs are used indicates the presence of multiple nucleoside analog resistant strains or mutations.