Abstract: Global fitness analysis makes use of a genomic library of tagged deletion strains. We used this approach to study the effect of chitosan, which causes plasma membrane stress. The data were analyzed using T-profiler, which was based on determining the sensitivity to chitosan of groups of deletion strains, as defined by Gene Ontology (GO) and by genomic synthetic lethality screens, in combination with t-statistics. The chitosan-hypersensitive groups included a group of deletion strains characterized by a defective HOG signaling pathway, indicating that the HOG pathway is required for counteracting chitosan-induced stress. Consistent with this, activation of this pathway in wild-type cells by hypertonic conditions offered partial protection against chitosan, whereas hypotonic conditions sensitized the cells to chitosan. Other chitosan-hypersensitive groups were defective in RNA synthesis and processing, actin cytoskeleton organization, protein N-glycosylation, ergosterol synthesis, endocytosis, or in cell wall formation, predicting that these cellular functions buffer the cell against the deleterious effect of chitosan. These predictions were supported by showing that tunicamycin, miconazole, and staurosporine (which target protein N-glycosylation, ergosterol synthesis, and the cell wall integrity pathway, respectively) sensitized yeast cells to chitosan. Intriguingly, the GO-defined group of deletion strains cytosolic large ribosomal subunit was more resistant to chitosan. We propose that global fitness analysis of yeast in combination with T-profiler is a powerful tool to identify specific cellular processes and pathways that are required for survival under stress conditions.