Aim: To compare the difference between the measured and target values in vancomycin external quality assessment (EQA) samples and to investigate the factors for underestimating its concentration. Materials & methods: A retrospective analysis of 195 international vancomycin EQA results was performed. Deviations of the concentrations determined by TDx fluorescence polarization immunoassay (FPIA), Axsym FPIA and Architect chemiluminescence microparticle immunoassay (CMIA) method were -2.43, -16.28 and -10.53%, respectively. Chromatographic peaks of the crystalling degradation products appeared in samples with large deviations. Vancomycin were degraded after long-term transporting and high temperature. Conclusion: Vancomycin concentrations measured by Axsym FPIA and Architect CMIA methods were likely to be underestimated. Long-term transporting resulted in low EQA results, suggesting that establishing a local EQA system for vancomycin is essential.