Background: Opioids, cannabis and nicotine are the oldest and highly abused substances globally. The detection and identification of these drugs is a major challenge for the law enforcement agencies as well as forensic chemists, especially when the cases pertain to sexual assaults/drug-facilitated sexual assaults. Aim: To develop a determination method which can quantify opioids, cannabinol and nicotine simultaneously in whole blood. Objective: This paper presents a validated method for the identification and quantitation of Morphine (MOR), Codeine (COD), Thebaine (THB), Papaverine (PAPA), Cannabinol (CBN) & nicotine (NIC) using High performance thin layer chromatography- Mass spectrometry HPTLC-MS, from whole blood at postmortem. Material & Method: Quantitative analysis of MOR, COD, THB, PAPA, CBN and NIC were done using HPTLC and MS was operating in selective ion-monitoring mode for accurate identification of the drugs under study. Small Volume Liquid  extraction (SVLE) technique using ethyl acetate-hexane (80:20) were used for the extraction of blood. Pre-coated HPTLC (silica gel G 60 F254) plates were developed using mobile phase ethyl acetate: methanol: ammonia (8.5:1:0.5). Result: The screening of HPTLC plates was done by UV light and the m/z ratio of drugs was obtained by lifting spots from plates using the HPTLC-MS interface. Method validation was done according to the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH) guidelines. The technique will encourage forensic chemists to embrace the validated HPTLC-MS method.  Conclusion: The developed HPTLC-MS method is simple, sensitive, precise, accurate, economic and can be used for quantitative analysis of MOR, COD, THB, PAPA, NIC and CBN in blood.