In this work, the application of matrix-assisted laser desorption ionization (MALDI)-quadrupole time-of-flight (QTOF)-tandem mass spectrometry (MS/MS) to the quantitation of the anticholinergic/antiparkinson drug biperiden in human whole blood, and the estimation of new phase I metabolites of biperiden in human urine, were performed for the first time. The limit of detection of biperiden in human whole blood was 0.3 ng/mL with a quantitation range of 1–100 ng/mL using 20 μL each of whole blood. The developed method was successfully applied to the determination of biperiden in whole blood obtained from two deceased subjects having a history of biperiden treatment. The concentrations of biperiden in the whole blood samples were 1.7 and 17.5 ng/mL. The MALDI-QTOF-MS/MS method was also used to estimate the metabolites of biperiden in urine obtained from the subjects. Six new metabolites formed by hydroxylation and/or dehydration were detected.