In this study we aimed to analyze the protein composition of the urine collected from the healthy animals and compare it to the two diabetic groups (DM I normoalbuminuric diabetic dogs; DM II diabetic dogs with microalbuminuria). We tried to identify potential urinary proteins which could be up- or downregulated in diabetic patients even before the appearance of microalbuminuria. Methods: After obtaining urine, we performed two-dimensional electrophoresis, followed by Delta2D software analysis, which allowed for selection and identification with MALDI-TOF spectrometry, statistically significant differentially expressed proteins. Our study revealed 286 common protein spots on 2D gels from the diabetic and control group. From these proteins five were positively identified by MALDI-TOF MS. To further evaluate the five differentiating proteins, the Panther program was used to assign them to appropriate biological process. Conclusion: Significant number of identified proteins play a role in intracellular signaling—vesicle formation, bonding, transport through membranes. This may suggest that first signs of kidney diabetic cellular impairment may be seen in the urine composition before any clinical signs occur.