The analysis of exhaled breath has attracted considerable interest for use in disease diagnosis and for monitoring treatments. Mass spectrometry (MS)-based approaches have been proven to be powerful analytical tools for breath analysis in clinical applications such as disease research, diagnosis, and monitoring. This review highlights MS methodologies as potential clinical tools in diagnosing human diseases. Different breath sampling methods and MS methods for breath analysis are reviewed, emphasizing their features, advantages, and limitations. Breath biomarkers from different diseases are summarized, describing their specificity, sensitivity, and drawbacks. The future perspectives and challenges on further development of MS-based breath diagnostics are discussed.