The bacterial formation of trimethylamine (TMA) has been linked to cardiovascular disease. This review focuses on the methods employed to investigate the identity of the bacteria responsible for the formation of TMA from dietary choline and carnitine in the human gut. Recent studies have revealed the metabolic pathways responsible for bacterial TMA production, primarily the anaerobic glycyl radical-containing, choline-TMA lyase, CutC and the aerobic carnitine monooxygenase, CntA. Identification of these enzymes has enabled bioinformatics approaches to screen both human-associated bacterial isolate genomes and whole gut metagenomes to determine which bacteria are responsible for TMA formation in the human gut. We centre on several key methodological aspects for identifying the TMA-producing bacteria and report how these pathways can be identified in human gut microbiota through bioinformatics analysis of available bacterial genomes and gut metagenomes.