Chirality is defined as the property of a molecule to exist in two non-superimposable mirror images, known as enantiomers. Despite the presence of numerous physical properties in common, enantiomers exhibit divergent interactions with chiral biological systems, thereby impacting pharmacological activity and toxicity. The thalidomide case represented a significant turning point in the realm of pharmaceutical regulation, underscoring the paramount importance of enantiomeric separation for ensuring drug safety. The advent of sophisticated techniques, such as chiral HPLC in conjunction with mass spectrometry, has rendered the analysis of complex chiral samples with exceptional precision attainable. This analysis is of crucial importance not only for research and clinical practice, but also for forensic science and chemical and forensic intelligence. It allows us to distinguish between legal and illegal uses, identify routes of administration, metabolic processes and synthesis methods.