Analysis of vitreous urea nitrogen (VUN) and creatinine (CREA) has been shown to be a useful indicator of azotemia in human postmortem examinations. Previous studies in cats, dogs, horses, and cattle have shown a good correlation between serum and postmortem ocular fluid for urea and creatinine. The aim of this study was to evaluate differences in VUN, CREA, and magnesium (MG) concentrations in vitreous humor collected postmortem from cats that presented with renal or post-renal disease and cats without renal or post-renal disease. Nine cats with renal or post-renal disease and twenty cats without renal or post-renal disease that underwent autopsy were used in this study. Collection of postmortem vitreous humor was performed, and vitreous samples were frozen at −80 °C for a minimum of 24 h prior to analysis using an in-clinic dry chemistry analyzer. Overall, there were statistically significant differences for VUN, CREA, and MG between cats with renal or post-renal disease and cats without renal or post-renal disease. Analysis of vitreous humor from cats suspected to have renal or post-renal disease can provide useful diagnostic information pertaining to renal function and issues in the urethra and urinary bladder. Future studies exploring postmortem vitreous chemistry in cats are warranted.