Driven by the advent of metabolomics, recent years have seen renewed interest in the investigation of yeast metabolism. Here we provide a practical guide to metabolomic analysis of yeast using liquid chromatography-mass spectrometry (LC-MS). We begin with background on LC-MS and its utility in studying yeast metabolism. We then describe key issues involved at each step of a typical yeast metabolomics experiment: in experimental design, cell culture, metabolite extraction, LC-MS, and data processing and analysis. Throughout, we highlight interdependencies between the steps that are relevant to developing an integrated workflow which effectively leverages LC-MS to reveal yeast biology.