At the moment, technologies based on the use of nanomaterials in various fields of medicine and industry are developing. The study of the influence of metal nanoparticles on microorganisms is an important area of modern biotechnology and microbiology. Of particular interest in this regard, is the use of microbiological methods for assessing the effects of metal nanoparticles on living organisms. Among other nanoparticles, zinc oxide nanoparticles are probably the most widely used nanomaterials. The cell viability is one of the important parameters for the determination of nanotoxicity. The present research paper contains information related to the validation of the method for microbial counting for pigmented yeasts cultivated in the presence of ZnO nanoparticles. The validation of this method was performed according to the USP 30-NF25 and method validation by U.S. Environmental Protection Agency. Pigmented yeasts were selected for this research. The microbial counting method was validated to ensure precision, accuracy, linearity and robustness and can be applicated safely for the study of the effect of nanoparticles on microorganisms cell viability.