A capillary electrophoretic assay was developed to measure the ribonuclease (RNase) H activity of human immunodeficiency virus (HIV) type 1 reverse transcriptase. Cleavage of a fluorescein-labeled RNA-DNA heteroduplex was monitored by capillary electrophoresis. This new assay was used as a secondary assay to confirm hits from a high-throughput screening program. Since autofluorescent compounds in samples migrated differently from both substrate and product in most cases, the assay was extremely robust for assaying enzymatic inhibition of such samples, in contrast to a simple well-based approach. The assay was broadly applicable to other RNases H, specifically those from human, Escherichia coli, and HIV-2, although product profiles varied for each enzyme.