The assessment of pitching and cropping yeast quality is of importance in order to ascertain adequate fermentation performance. Viability and vitality are common terms used to describe the quality of yeast. Viability corresponds to the percentage of dead cells in a sample, whereas vitality represents the physiological state of the yeast. There are many methods available to assess the quality of a yeast sample; however most of these techniques require further development to reach the level of sensitivity and reproducibility necessary to assess yeast populations that exhibit physiological stress. Methylene blue remains an industry standard for viability assessment, even though the efficiency of this stain is highly controversial. It has been suggested that methylene violet might provide a greater accuracy of viability than methylene blue due to the occurrence of impurities in the latter; however viability assessment using brightfield methods remains subjective. The objective of this study was to identify an alternative to brightfield reductive dye techniques using fluorophores for the determination of viability. Three genetically distinct brewing strains were utilised for this study: a lager (L138), ale (NCYC2593) and cider (TC16) strain. Viability and vitality studies were performed on yeast cell populations under a variety of specific stress conditions. Cohorts of cells were submitted to osmotic, ethanol, starvation and oxidative stress. In addition cell populations exhibiting different viabilities, obtained from mixing heat-treated and stationary phase cells, were assessed for vitality. Viability was assessed using the fluorophore dyes; oxonol (DiBAC(4)), MgANS (8-anilino-1-naphtalene-sulfonic acid) and FUN1, and compared to conventional brightfield dyes such as methylene blue and violet. Vitality was evaluated using FUN1 and the level of fluorescence was assessed either microscopically or quantitatively using a fluorimeter with a microplate reader attachment. Vitality was also determined by measuring ATP levels using the luciferin-luciferase reaction and by recording the subsequent fluorescence emitted. It was observed that Oxonol and MgANS successfully distinguished between live and dead cells without ambiguity, regardless of the yeast strain employed. These stains were perceived to be less subjective to the operator compared to brightfield dyes due to the lack of intermediate colour variations. It is suggested that fluorophore technology may represent a simple reproducible alternative to methylene blue. It was demonstrated that FUN1 might be a potentially good indicator of vitality rather than viability and the investigation of new techniques using fluorophores will lead to the development of simple, reliable and reproducible methods to assess yeast quality.