5-HT3 receptor antagonists, a class of potent antiemetic drugs, are often used clinically as the basis for prophylactic treatment of vomiting induced by cancer radiotherapy, anesthesia and postoperative period. Commonly used 5-HT3 receptor antagonists include ondansetron, granisetron and dolasetron. These pharmacological agents can be administered via various routes, including oral, intravenous, and intramuscular pathways. It is crucial to acknowledge that although 5-HT3 receptor antagonists are highly efficacious in managing nausea and vomiting, they may also induce side effects such as headache, constipation, or diarrhea. Furthermore, improper use or overdose can potentially impact the nervous system. To mitigate the adverse effects associated with this class of drugs and to establish the optimal dosage, it is essential to accurately extract and assay 5-HT3 receptor antagonists. In this review, we overviewed the pretreatment techniques used for the extraction and purification of 5-HT3 receptor antagonists, as well as the identification and quantification methods. The common pre-treatment methods for 5-HT3 receptor antagonists include ultrasonic-assisted extraction, liquid-liquid extraction, solid-phase extraction, solid-phase microextraction and dispersive solid-phase microextraction. Techniques such as high-performance liquid chromatography-mass spectrometry, high-performance liquid chromatography-coupled with other detectors like ultraviolet and fluorescence, and electrochemical sensors are extensively utilized in the detection and quantification of 5-HT3 receptor antagonists. In addition, latest advances in the techniques were summarized, detailing the features and advantages of each approach.