Recently, there has been an increase in the use of several nephrotoxicity biomarkers in preclinical experiments. In addition, it has been indicated that the result may have been influenced by secondary factors, such as sample storage condition or storage period. In this study, we have assessed the variation in urinary nephrotoxicity biomarkers as a result of urine storage conditions and storage period of the urine. Urine was sampled from specific pathogen-free Sprague-Dawley rats (19 weeks old), which were housed individually in hanged stainless steel wire mesh cages. Urine was stored at $20^{circ}C$, at $4^{circ}C$, or at $-70^{circ}C$ after sampling. The levels of the biomarkers such as beta-2 microglobulin (B2M), cystatin-C (Cys-C), N-acetyl-${eta}$-D-glucosaminidase (NAG), micro albumin (MA), micro protein (MP) were measured at 6, 24, 48 and 144 hr after sampling. The B2M level was significantly decreased at 6, 24, 48, and 144 hr compared to 0 hr at $-70^{circ}C$ (p < 0.05, p < 0.01, p < 0.05, and p < 0.05, respectively) and 24 and 144 hr at $20^{circ}C$ (p < 0.01, p < 0.01, respectively). The Cys-C level was significantly decreased at 144 hr compared to 0 hr at $4^{circ}C$ (p < 0.01), at $20^{circ}C$ (p < 0.05) and at $70^{circ}C$ (p < 0.01). MP and MA levels were not different for 144 hr in all storage conditions. Taken together, B2M and Cys-C levels were modulated by storage temperature and period. For the enhancement of test accuracy, it is suggested that strict protocols be established for samples to minimize the effects of the storage conditions on the detected levels of biomarkers.