Expression of human endogenous retroviruses (HERV) has been recurrently observed during cellular differentiation or transformation processes in both cell culture and in vivo. Quantitative approaches that analyze variations in HERV transcription could therefore be valuable for cancer diagnosis. We have developed a quantitative assay combining multiplex degenerate PCR (MD-PCR) and a colorimetric Oligo Sorbent Array (OLISA). Quantification of the expression of these multifamily genes relies on the optimization of the amplification primer mix, that is, the primer degeneracy, the relative concentration of each primer and the total amount of primer. Amplification products of each of the nine studied HERV families are independently and specifically detected and quantified using the OLISA microarray. This method constitutes an improvement over previous pan-retrovirus amplification-based methods, which are mainly qualitative. Furthermore, as MD-PCR/OLISA simultaneously monitors several HERV families, it challenges single-family quantitative RT-PCR. Last, the protocol below provides general rules for the design of MD-PCR applications. Once primers have been designed and optimized, the procedure can be completed in 2 days.