Commonly used clinical indicators of renal disease are either insensitive to early dysfunction or have delayed results. Decreased glomerular filtration rate (GFR) indicates renal dysfunction before there is a loss of 50% of functional nephrons. Most tests evaluate global rather than individual kidney function. Dynamic computed tomography (CT) and Patlak plot analysis allows for individual GFR to be tested. Our objectives were to establish a procedure and provide reference values for determination of global GFR in 10 healthy cats using dynamic CT (CTGFR). This method of GFR determination was compared against serum iohexol clearance (SIC). A single CT slice centered on both kidneys and the aorta was acquired every fifth second during and after a bolus injection of iohexol (240 mgI/ml; 300 mgI/kg) for 115 s. Using data from this dynamic acquisition, Patlak plots were obtained, GFR was calculated, and results were compared to global GFR determined by iohexol clearance. The average global CTGFR estimate was 1.84 ml/min × kg (SD = 0.43; range = [1.22, 2.45]). The average global GFR measured using SIC was 2.45 ml/min × kg (SD = 0.58; range = [1.72, 3.69]). GFR measurements estimated by both dynamic CT and SIC were positively associated (estimated Spearman rank correlation coefficient = 0.72; P = 0.0234). The CTGFR method consistently underestimated GFR with a bias of −0.62 (SE = 0.1307) when compared to SIC (P = 0.0011). In healthy cats, CTGFR was capable of determining individual kidney function and appears clinically promising.