The objective of this study was to demonstrate that ribosomal RNA-targeted techniques could be used to quantify viable microorganisms when determining disinfection efficacy. The authors have developed a new technique to simultaneously measure ribosomal RNA as well as precursor ribosomal RNA, a key intermediate in the production of ribosomes. Ribosomal RNA and precursor ribosomal RNA were measured simultaneously in cultures of Acinetobacter calcoaceticus^T and Mycobacterium mucogenicum. The results demonstrate that precursor ribosomal RNA is a sensitive measure of viability. The authors believe this approach holds tremendous promise for verifying disinfection efficacy for microorganisms using molecular biology-based monitoring. In the future, coupling the rapid developments in molecular biology-based technology with the critical measurement of cellular viability via detection of ribosome genesis may change the way disinfectants are evaluated. Includes 12 references, figures.