OBJECTIVE A new assay, much more rapid and efficient than the existing standardized tests, is introduced for the evaluation of bactericidal activity of chemical disinfectants and antiseptics under simulated practical conditions of use. RESULTS The bactericidal activity of biocides was quantified using a novel semi-automated assay based on the European Norm (EN) standard suspension tests but determining bacterial cell viability by intracellular adenosine tri-phosphate (ATP) content quantification instead of traditional culture-based microbiological techniques. The new test was validated by comparison to the standard suspension tests EN 1276 and EN 13727. During the validation, the linearity of the ATP detection system, limit of detection, specificity, sensitivity, relative accuracy and precision (repeatability and reproducibility) were determined. CONCLUSIONS The validation study showed that the new assay evaluates the activity of biocides as well as the EN standard suspension tests, but it allows a large number of test conditions to be efficiently analysed. CONCLUSIONS The new test can therefore be applied to accurately establish the lowest active concentration (MBCs) of disinfectants or antiseptics under simulated practical conditions of use and to compare the susceptibility of a large number of strains and conditions via inactivation curves. This is not possible in any reasonably practicable way with the EN standards considering the time and cost required for each determination.