Tramadol is a synthetic opioid drug used in the treatment of chronic and acute pain. An abnormal prevalence of its misuse in elite sport to overcome pain resulting from prolonged physical effort was recently reported. However, besides its antinociceptive effects, tramadol consumption is associated with negative effects such as numbness, confusion, and reduced alertness. This fact prompted the Union Cycliste Internationale to ban the use of tramadol in cycling competitions. Herein, we present the development of a dried blood spot (DBS) sample collection and preparation method followed by a liquid-chromatography mass spectrometry (LC-MS) analysis to rapidly determine the presence of tramadol and its two main metabolites in blood samples. The detection window of each analyte was evaluated and the analysis of performance on various MS platforms (HRMS and MS/MS) was assessed. Tramadol and its two main metabolites were detected up to 12 h after the intake of a single dose of 50 mg of tramadol in positive controls. In professional cycling competitions, 711 DBS samples collected from 361 different riders were analysed using the developed methodology, but all returned negative results (absence of parent and both metabolite compounds). In the context of professional cycling, we illustrate a valid method bringing together the easiness of collection and minimal sample preparation required by DBS, yet affording the performance standards of MS determination. The proposed method to detect tramadol and its metabolites was successfully implemented in cycling races with a probable strong deterrent effect.