Abstract Background Measurement of direct oral anticoagulants (DOACs) concentration in patient blood is essential in special clinical circumstances. Methods We developed a fast, selective and sensitive method for simultaneous measurement of DOACs in human plasma consisting of an automated online solid-phase extraction method coupled with ultra-performance liquid chromatography electrospray ionization-tandem mass spectrometry (online SPE-UPLC-MS/MS). Results The calibration curves of all DOACs were linear over the working range (apixaban: 0.25–760 μg/L, r > 0.99; dabigatran: 0.5–900 μg/L, r > 0.99; edoxaban: 0.6–800 μg/L, r > 0.99; rivaroxaban: 0.5–900 μg/L, r > 0.99). Limits of detection in the plasma matrix were < 0.2 μg/L, whereas the lower limits of quantification were < 0.6 μg/L for all DOACs. The intraassay and interassay CV for all DOACs were < 6%. Mean recoveries were between 61.4% and 91.6%. Method comparison between our online SPE-UPLC-MS/MS assay and commercially available functional based coagulation assays using patient samples showed a high degree of correlation for all investigated DOACs. Conclusions We developed and validated the first online SPE-UPLC-MS/MS method for fast, sensitive, specific, and reliable measurement of the new generation of DOACs and compared this method with commercial available coagulation assays. Highlights • A highly specific automated mass spectrometric method to measure four direct oral anticoagulants (DOACs) was developed and fully validated. • The total method run time was only 4 min using automated online SPE-UPLC-MS/MS. • Effective sample preparation was performed by a combination of protein precipitation with an internal standard mixture and an online SPE step. • Fast, specific, and sensitive measurement of DOACs in plasma was allowed. • Mass spectrometric measurements of DOACs were compared with commercially available functional based coagulation assays. [ABSTRACT FROM AUTHOR]