OBJECTIVES: Measuring serum 25-hydroxyvitamin D is key in clinical labs, but inter-laboratory variations risk diagnostic errors. This study evaluates the performance of current in-house liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods used in top Chinese clinical laboratories and proposes an optimized method for improving serum 25(OH)D measurement accuracy and reliability. METHODS: Seven serum pools with different concentrations of 25(OH)D were prepared and sent to 12 participating laboratories for multiple repeat analysis with their current in-house LC-MS/MS methods and then an optimized LC-MS/MS method. Precision was assessed in terms of coefficient of variance (CV), and trueness was assessed in terms of bias referring to the U.S. National Institute of Standards and Technology (NIST) reference measurement procedure (RMP). The analytical performances of the two methods were compared and evaluated. RESULTS: Eighty percent and 90 % of the laboratories achieved the defined performance criteria (CV, <
12.5 %
mean bias, <
8.3 %) with the optimized method for the measurement of 25(OH)D2 and 25(OH)D3, compared with 43 % and 57 % of the laboratories meeting the criteria with their in-house methods, respectively. Precision and trueness improved after applying the optimized method. Although the optimized method didn't not ensure that all laboratory samples meet the measurement uncertainty (MU) criteria (MU<
13.6 %), particularly for low-concentration samples, it significantly reduced the MU compared to the in-house method. CONCLUSIONS: Precision, trueness and MU improved after applying the optimized method. Nonetheless, more efforts are needed to ensure the reliability and accuracy of 25(OH)D measurements in clinical laboratories in China.