Random mutagenesis provides a valuable tool to elucidate gene function and to draw connections between gene products and molecular pathways. This chapter provides two protocols for the random mutagenesis of Komagataella phaffii, along with methods to screen for the resulting mutant phenotypes. Genes are first disrupted by the insertion of a non-homologous DNA cassette. In the next step, primers designed to anneal to the cassette enable the identification of the mutagenized locus. We applied our system in K. phaffii to enhance recombinant protein secretion of horseradish peroxidase (HRP) and β-galactosidase, respectively. The subsequent screening identified unexpected genes that were later confirmed to benefit recombinant protein secretion, validating the substantive potential of the methodology.