High-throughput chemical genomics uses phenotypic profiling of strain libraries under defined chemical and environmental conditions to identify gene functions. This approach enables the mapping of biological pathways and can potentially highlight drug targets. Chemical genomic data sets have been springboards for numerous hypothesis-driven research projects, with direct implications for antimicrobial resistance and clinical outcomes. High-throughput phenotypic profiles are valuable tools for enriching microbial sequence data with functional annotations and benefiting the broader scientific community. This work provides a step-by-step guide for conducting chemical genomics screens from start to finish.