The utility of microalgal biomass and bioproducts depends on long-term maintenance of certain physiological or biochemical features of the species. While unique characteristics may not be durably maintained with general subculture, cryopreservation methods better prevent alterations from desired characteristics. Post-thaw viability is critical to establishing microalgal cultures, and there is a critical need to effectively and rapidly evaluate microalgal viability after the post-thawing process. In the present study, we developed a rapid assay based on the change of fluorescence ratio to determine microalgal viability post-thaw. It was shown that the assessment of microalgal viability by the fluorescence ratio method correlated well with that of the FDA-staining (R-2 = 0.978) and regrowth method (R-2 = 0.976), demonstrating that the present method could be applied in the high throughput detection of viability of microalgal strains. Subsequent to establishing this method, we aimed to find out optimal cryopreservation protocol for each strain from a group of 125 microalgal strains. The viability of these strains under different treatments was quickly evaluated by the fluorescence ratio method. Of these strains, 95 attained post-thaw viability over 60%. DMSO was a suitable cryoprotectant for most strains at a concentration <= 10%. Based on the dataset, the relative contribution of 3 variables-genus, cryoprotectants and concentration to post-viability was analyzed with the Random Forest (RF) classification method. All variables together could explain 97.8% of the viability, and type and concentration of cryoprotectant could explain 59.1% in Chlorophyta. This study provided a new approach for viability assay and demonstrated that this method can facilitate to find, out the optimal protocols for cryopreservation of microalgal strains.