Drug abuse continues to be a major concern for public health and law enforcement agencies around the world. This requires development and application of reliable analytical techniques for accurate examination of abused drugs in various biological specimens. Blood, urine, hair and oral fluid are some of the routinely used biological specimens in forensic toxicology, each offering different advantages and limitations based on the purpose of analysis. Over the years, various analytical techniques have evolved from screening techniques to highly sensitive instrumental techniques. This literature review provides a summary of the routinely used techniques for the examination of drugs of abuse in biological specimens, particularly emphasizing on immunoassay, Gas Chromatography-Mass Spectrometry (GC-MS), Liquid Chromatography-Mass Spectrometry (LC-MS) and High-resolution Mass Spectrometry (HRMS). This review discusses principles, applications, strengths, limitation and recent advancement of these techniques, along with suitability of different biological samples for forensic toxicological examination. Comparative evaluation of these analytical techniques shows us how to make drug detection more accurate, sensitive and reliable.