This report describes the development of a one-tube multiplex reverse transcriptase (RT)-PCR assay for the simultaneous detection of hepatitis C virus (HCV) and human immunodeficiency virus (HIV) in plasma samples. The assay was evaluated with two panels of HCV- and HIV-1-positive samples, as well as negative plasma specimens. Extraction and amplification of HCV and HIV-1 RNA from plasma samples were performed in a single reaction, and amplified genomes were detected with specific probes. Serial dilutions of the HCV and HIV-1 first World Health Organization International Standards were used to evaluate the sensitivity of the method. Two RNA controls were constructed to determine inter-assay variations and the sensitivity of the amplification step. The assay had good specificity and detected all the genotypes and subtypes tested. The analytical sensitivity of the entire assay was 100 IU/mL for HCV and 200 IU/mL for HIV-1, while the amplification step detected ten copies/reaction for HCV and 20 copies/reaction for HIV-1. The multiplex assay allowed the simultaneous extraction, amplification and detection of two virus genomes, thereby providing an important practical advantage and an efficient approach for analysing individual and pooled plasma donations.