The importance of in-depth knowledge about the pharmacokinetics of a drug is evident because pharmacokinetic behaviour may correlate with activity and toxicity. The most elaborate pharmacokinetic investigation is a so-called mass balance study employing a radioactive tracer. A mass balance study investigates the plasma pharmacokinetics and excretion of both the unchanged drug and the total radioactivity (drug and metabolites), and allows elucidation of the metabolic fate of a drug. The main objective is the maximum recovery of the radioactive dose in urine and faeces. However, there is little concrete guidance on how to perform or assess such a mass balance study. Based on a critical review of the available literature and regulation, we discuss the design, conduct and evaluation of mass balance studies. The study must be preceded by pre-clinical studies to optimise the design. Compliance with available regulation (US FDA is more specific than European guidelines), choice of radioisotope (tritium may be lost and is consequently used less often), selection of subjects and safe administration of a relevant dose are critical. The drug (both the labelled and the non-labelled portion of the dose) must be prepared, characterised and administered, and the biological samples must be collected and analysed properly to obtain reliable and meaningful results. A recent development is the use of highly sensitive accelerator mass spectrometry for (14)C-detection, which allows the use of much lower amounts of radioactivity. Calculating the mass balance requires quantification of any possible losses of radioactivity during the study, e.g. during administration (adsorption to tubing). Total recovery should be at least 90% of the administered dose. A lower recovery, which is not uncommon, must be explained by biological factors such as a long decay half-life, irreversible binding to tissue components, or loss through expiration. The extent and relevance of metabolism is determined by comparing the pharmacokinetics of the unchanged drug and the total radioactivity. Metabolic profiling supplements existing knowledge of metabolism. We illustrate the important aspects of a mass balance study with literature on anticancer drugs. This review could serve as guidance for future mass balance studies.