The requirements on initial testing in doping control are getting tighter regarding efficiency and speed while the scope of analytes is getting more diverse and, consequently, the need for high-throughput methods is apparent. In this study, a comprehensive screening method for doping agents in human urine is presented, based on solid phase extraction (SPE) and liquid chromatography-time-of-flight mass spectrometry (LCTOFMS). The method covers most of the compound groups in the list of prohibited substances by World Anti-Doping Agency (WADA). Mixed-mode SPE on two types of sorbent and the use of negative ionization mode besides the commonly used positive mode in electrospray ionization (ESI) allowed detection of acidic compounds, such as sulpho-conjugated metabolites. A run time of 8 minutes for each of the two ESI polarities was achieved. The method was validated regarding relative ionization efficiency, selectivity and signal to noise at the WADA's minimum required performance limit (MRPL) level, resulting in the acceptance of 197 compounds. A selection of 20 compounds was submitted for a more thorough validation, including extraction recovery, repeatability and linearity. Recovery and linearity (R(2)) varied mainly between 83-115% and 0.78-0.99, respectively. Median values for repeatability at the MRPL and 10 x MRPL levels were below 20%. A mean and median mass accuracy of 1.2 and 0.80 mDa, respectively, was achieved. The present method represents at the moment the widest coverage of low molecular weight prohibited substances for the screening in sports, providing an approach for further rationalisation of the analytical work-flow in the doping control laboratories.