Novel psychoactive substances (NPS) are becoming increasingly popular worldwide in recent years, some of which have been reported to cause considerable harm and even fatalities. Currently, simultaneous screening for a comprehensive panel of conventional and novel drugs of abuse is not widely available in most clinical laboratories. The aim of this study was to establish a chromatography/mass spectrometry-based analytical system for the simultaneous detection of conventional drugs of abuse and NPS in urine. Sample preparation entails enzyme digestion and solid phase extraction; analytes were then detected by liquid-chromatography tandem mass spectrometry (LC-MS/MS) with multiple reaction monitoring. Forty-seven conventional drugs (28 parent drugs, 19 metabolites) and 46 NPS analytes (44 parent drugs, two metabolites) are covered by the established method, which has been validated according to international guidelines. The method was then applied to 964 urine samples collected from drug abusers and the results revealed the presence of two NPS - TFMPP and methcathinone - as well as conventional drugs of abuse. To conclude, an LC-MS/MS method has been established that allows the simultaneous detection of over 90 conventional as well as novel psychoactive substances and metabolites in urine samples. The method was successfully applied to authentic specimens revealing the presence of conventional as well as novel drugs of abuse in the local population.