Direct oral anticoagulants (DOACs) are increasingly being used, primarily due to their drug stability and patient convenience. Although these drugs have been evaluated to be well tolerated in numerous clinical trials, their impact on in-vivo anticoagulation effect, variability and therapeutic drug level remains unknown. Hence, we aim to study the effect and variability of DOACs on thrombin generation via the calibrated automated thrombogram. Anonymized coagulation specimens from outpatients on warfarin were collected. Pooled normal plasma samples were spiked with increasing concentrations of dabigatran, rivaroxaban and apixaban. Similarly, plasma samples with normal coagulation profiles were spiked with two concentrations each of dabigatran, rivaroxaban and apixaban. Thrombin generation via calibrated automated thrombogram was run using the above samples and compared with a dataset of normal controls. Increasing international normalized ratio was associated with a reduction of endogenous thrombin potential (ETP) and thrombin peak and increasing lag time. Factor Xa inhibitors produced a flattened thrombin generation curve with a reduction of thrombin peak and relative preservation of ETP. Direct thrombin inhibitors produced a reduction of both ETP and thrombin peak and increasing lag time. Seventy-one citrated plasma samples (26 dabigatran, 21 rivaroxaban and 24 apixaban) were evaluated. The two concentrations produced a reduction of thrombin generation parameters with significant interindividual variability compared with neat plasma of 35–93, 13–31 and 18–71% and for dabigatran, rivaroxaban and apixaban, respectively. Thrombin generation can measure the anticoagulation effect of commonly used DOACs, with each drug having a unique thrombin generation profile. The variability noted using the same concentration suggests significant interindividual pharmacodynamic differences, which maybe relevant with respect to efficacy as well as bleeding side effects. Further delineation of the modifiers of interindividual differences is required in the in-vivo setting.