At the time of writing, this work appears to be the first published report on the coupling of on-line solid phase extraction (SPE) with ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) for the simultaneous detection of nicotine, cotinine and trans-3'-hydroxycotinine in human urine. The advantages of both on-line SPE (speed, automation, less labor intensive) coupled with UPLC-MS/MS (speed, sensitivity) offer a viable option for efficient and economical biomonitoring studies for the assessment of active and passive exposure to tobacco smoke and clinical studies focusing on smoke cessation techniques. In a first approach, a 1:100 dilution of the urine was applied to screen for both passive and active exposure. Intra- and inter-batch reproducibility of this fast method (10min) was assessed for three concentration levels and were found to be less than 8% for each analyte while the accuracy was between 89 and 113%. To further improve the sensitivity of this approach when focusing only on passive smokers, the performance of a 1:10 dilution with the on-line SPE UPLC-MS/MS system was also tested. Results indeed show better sensitivity (LOQ's 1.0, 1.0 and 5.0μg/l for respectively cotinine, nicotine and trans-3'-hydroxycotinine) and good analytical performance for all other analytical parameters on the low levels tested here. Both methods were applied to measure the concentration of nicotine, cotinine and trans-3'-hydroxycotinine in the urine of 53 volunteers (smokers and non-smokers) recruited via an internal call at the Scientific Institute of Public Health. For non-smokers and smokers, cotinine levels e.g. were respectively between 1.0-470μg/l and 97-2381μg/l. Both these on-line SPE UPLC-MS/MS methods showed their potential for dedicated future large biomonitoring projects as they made it possible to analyze large series of samples in a fast, sensitive, robust and cost-efficient manner.