OBJECTIVE To investigate effects of storage conditions on the canine urine protein-to-creatinine ratio (UPC) and on SDS–agarose gel electrophoresis (AGE) of urinary proteins. SAMPLE Urine specimens from 20 proteinuric (UPC > 0.5) and 20 nonproteinuric (UPC ≤ 0.2) dogs. PROCEDURES UPC and SDS-AGE were performed on urine specimens stored at room temperature (20°C) and 4°C for up to 5 days and at −20° and −80°C for up to 360 days; some specimens were subjected to 3 freeze-thaw cycles. Results were compared with those obtained for fresh urine specimens. RESULTS UPC was not affected by storage at room temperature or by freezing. A decrease in UPC was observed for specimens from nonproteinuric dogs after 5 days at 4°C (10%) and from both groups after 90 days at −20° and −80°C (≤ 20% and ≤ 15%, respectively). The SDS-AGE profiles revealed no visual changes regardless of duration of storage for specimens stored at room temperature, 4°C, and −80°C, except for 1 profile after 360 days at −80°C. Repeated freeze-thaw cycles did not affect SDS-AGE profiles. Appearance or strengthening of high-molecular-weight bands that could alter interpretation was evident in SDS-AGE profiles after storage at −20°C for ≥ 15 days (31/40 dogs). CONCLUSIONS AND CLINICAL RELEVANCE Storage of urine at −20° or −80°C for up to 1 year influenced the UPC without affecting clinical interpretation. Storage of urine specimens at −20°C impaired visual analysis of SDS-AGE. When SDS-AGE cannot be performed on fresh or recently refrigerated urine specimens, storage at −80°C is recommended.