Random transposon mutagenesis allows for relatively rapid, genome-wide surveys to detect genes involved in functional traits, by performing screens of mutant libraries. This approach has been widely applied to identify genes responsible for activities of interest in multiple eukaryote and prokaryote organisms, although most studies on microorganisms have focused on pathogenic and clinically relevant bacteria. In this chapter we describe the implementation of an in vitro Tn5-based transposome strategy to generate a large collection of random mutants in the gut commensal Bifidobacterium breve UCC2003, and discuss considerations when applying this mutagenesis system to other Bifidobacterium species or strains of interest.