Bioanalysis of an endogenous compound such as leucovorin is never an easy task on a liquid chromatography tandem mass spectrometer (LC–MSMS). Unless it is necessary, regulatory guidance discourages working with surrogate matrices for calibration curve standard preparation. Herein, a selective and sensitive liquid chromatography–tandem mass spectrometry method for simultaneous determination of leucovorin and 5‐methyl tetrahydrofolic acid in human plasma was developed and validated. Stable labeled internal standards, i.e. leucovorin D4 and 5‐ methyl tetrahydrofolic acid 13C5, were used as internal standards to track and compensate the parent compounds during processing and extraction from plasma. The method involves a rapid solid‐phase extraction from plasma followed by reverse‐phase gradient chromatography and mass spectrometry detection with a total run time of 5 min. The method was developed and validated from 5 to 2,202 ng/ml for leucovorin and from 5 to 1,300 ng/ml for 5‐methyl tetrahydrofolic acid. The mean recoveries for leucovorin and 5‐methyl tetrahydrofolic acid were 100.4 and 100.9% respectively. The validated method enabled the simultaneous analysis of leucovorin and 5‐methyl tetrahydrofolic acid in samples from clinical pharmacokinetic studies of leucovorin. The peak concentrations of leucovorin and 5‐methyl tetrahydrofolic acid were 651–883 and 518–635 ng/ml, respectively, in fasted and fed conditions. The terminal half‐life values for leucovorin and 5‐methyl tetrahydrofolic acid were 9.3–10.5 and 9.2–17.6 h, respectively.