Inborn errors of metabolism (IEMs) are rare diseases caused by a defect in a single enzyme, co-factor, or transport protein. For most IEMs, no effective treatment is available and the exact disease mechanism is unknown. The application of metabolomics and, more specifically, tracer metabolomics in IEM research can help to elucidate these disease mechanisms and hence direct novel therapeutic interventions. In this review, we will describe the different approaches to metabolomics in IEM research. We will discuss the strengths and weaknesses of the different sample types that can be used (biofluids, tissues or cells from model organisms; modified cell lines; and patient fibroblasts) and when each of them is appropriate to use.