One-point (linear through zero) calibration is often used as a compromise between necessary calibration, workload, and time. The aim of the present study was to systematically check the applicability of one-point calibration by comparing bias and precision data obtained with full and one-point calibration. Data from validation studies of six mass spectrometry-based multianalyte bioanalytical assays were used for this purpose. Bias and intermediate precision datasets of full multiple-point calibration were compared to six one-point calibration datasets (A-F in rising calibrator concentration order) calculated from the same raw data. The datasets were statistically compared using the Friedman test followed by Dunn's multiple comparison test. The results obtained with full calibration and the different one-point calibrations were found to differ significantly (P < 0.05) in all of the six studied methods. The best one-point calibration results were obtained with calibrator D, with which acceptance criteria for bias and precision were fulfilled for the majority of analytes. However, some extremely high bias and precision data were obtained for some analytes in the low-concentration range. In conclusion, one-point calibration with a calibrator close to the center of the full calibration range can be a feasible alternative to full calibration.