Centrifugal elutriation discriminates cells according to their sedimentation coefficients, generating homogeneous samples well suited for genomic comparative approaches. It can, for instance, isolate G1 daughter cells from a Saccharomyces cerevisiae unsynchronized population, alleviating ageing and cell-cycle biases when conducting genome-wide/single-cell studies. The present report describes a straightforward and robust procedure to determine whether a cell population of virtually any yeast species can be efficiently elutriated, while offering solutions to optimize success. This approach was used to characterize elutriation parameters and S-phase progression of four yeast species (S. cerevisiae, Candida glabrata, Lachancea kluyveri and Pichia sorbitophila) and could theoretically be applied to any culture of single, individual cells.