Abstract: Objective To explore the method of preparing urine quality control samples in metabonomics research. Methods Urine samples collected from healthy volunteers and cadmium exposure individuals, and quality control samples were analyzed by LC/MS techniques. Then, the total ion currents, the content of endogenous metabolites and principal components were compared. Results Study samples consisted of 40 morning urine samples from female healthy volunteers, 40 morning urine samples from cadmium exposure females, and 18 quality control samples prepared by mixing 80 urine research samples at equal volume from 40 morning urine samples of female healthy volunteers and 40 morning urine samples of cadmium exposure females. The total ion currents of 6 samples randomly selected from healthy volunteers and 6 morning urine samples randomly from cadmium exposure females were disordered, but 6 quality control samples nearly overlapped in EST+/- conditions. The inter-quartile range of endogenous metabolites indicated that contents of quality control samples were significantly different from those of healthy volunteers and cadmium exposure females (both P＜0.05). The score plots of principal component analysis (PCA) showed that urine samples of healthy volunteers and cadmium exposure females distributed dispersedly, but quality control samples concentrated. Except five quality control samples used in balance system, the deviation of the other thirteen quality control samples was kept in 2SD.Conclusion Quality control samples prepared in this study could reflect the basic information of samples and the status of system control, and the method of preparing quality control samples in urine metabonomics research was established.