The metabolome contains all the biological end points of genomic, transcriptomic and proteomic perturbations, also including the influence of gut microbiota and the environment, giving a direct picture of an organism’s ongoing metabolic state. Metabolomics thus has the potential to be an effective tool for early diagnosis of disease, and also to be a predictor of treatment response and survival. In recent years, the development of instrumental systems has enabled more comprehensive coverage of the metabolome. Advances in mass spectrometry and chromatography have particularly improved both the efficiency of nontargeted metabolic profiling as well as the sensitivity and reliability of targeted analyses. Mass spectrometric techniques are also increasingly becoming accepted as a routine diagnostic tool in clinical laboratories. This review summarizes the most recent advances and current challenges in metabolomics, with a focus on mass spectrometric methods utilized in biomarker research, highlighted with selected examples.