Abstract <p>Antimetabolites, which are structural analogues of metabolites or carbon sources that exhibit toxicity by disrupting metabolism, can couple phenotypes of interest to growth and thereby facilitate the selection of relevant strains from large mutant pools generated by random mutagenesis. Toxicity and high costs of many antimetabolites provide incentives to minimise liquid volume during cultivation, while still capturing the genetic diversity in large mutant pools. This study aims to explore the use of high-throughput microbead-based cultivation and label-free enrichment of antimetabolite-resistant mutants. The generation of a large number (~ 10⁷ microbeads) of picolitre-sized cultivation compartments using water-in-oil emulsions enables high-throughput, parallel growth of millions of individual mutants in a total medium volume of just 0.3&#xa0;mL. A reduction in screening medium volume reduces the quantities of expensive antimetabolites required. Employing a fluorescence-activated microbead-sorting-based approach enables label-free screening and selection of mutants based on autofluorescence signals as indicators for growth. The potential of this microbead-based, label-free selection method was demonstrated by enriching a G418 (geneticin) antibiotic-resistant and a glucose analogue (2-deoxyglucose) resistant <i>Saccharomyces cerevisiae</i> strains by 16,000-fold and 600-fold, respectively, in single-step enrichment experiments.</p> Key points <p>• <i>Small-volume cultivation cuts screening costs for costly antimetabolite resistance</i>.</p> <p>• <i>Autofluorescence-based screening enables label-free detection of resistant mutants</i>.</p> <p>• <i>10</i>⁴- <i>and 10</i>³<i>-fold enrichment of antibiotic- and glucose analogue-resistant strains</i>.</p> Graphical Abstract <p></p>