Brucine and strychnine, primary bioactive constituents in traditional Chinese medicine, are extensively utilized in healthcare products due to their pharmacological and biochemical properties, including the enhancement of blood circulation, alleviation of cancer-related pain, and their anti-inflammatory and anti-angiogenesis effects. While brucine and strychnine possess indispensable pharmacological properties, they also pose serious toxicity and threats to human health. Consequently, it is imperative to develop reliable and sensitive methodologies for their pretreatment and quantification in various matrices. This comprehensive review encapsulates the prevailing pretreatment and analytical methodologies for brucine and strychnine in diverse samples from 2013 onwards. The routinely employed pretreatment techniques for target extraction and purification encompass liquid-liquid extraction, solid-phase extraction, and a range of microextraction methods. The determination strategies incorporate liquid chromatography-based methods, capillary electrophoresis methods, sensor methods, among others. Moreover, we have identified the strengths and weaknesses of each method and have forecast their future developmental prospects.