This protocol describes the creation of a site-saturation mutagenesis library for gene variant analysis. It begins with ordering site-saturation mutagenesis oligonucleotide pools from Twist Biosciences for each position in the gene of interest, excluding position 1. These pools, containing all possible synonymous and missense variants for each codon, are combined into tiled sublibraries to cover the entire gene sequence, with overlap to ensure completeness. This protocol enables the generation of a comprehensive mutant library for functional analysis of the gene, providing insights into the effects of various mutations. The process involves careful handling of materials, precise PCR and assembly steps, and thorough bacterial transformation and culture techniques.