Background: The purpose of this study was to compare the performance of the Cockcroft-Gault (CG), Modification of Diet in Renal Disease (MDRD-4), and Chronic Kidney Disease Epidemiology Collaboration (CKDEPI) equations for estimation of glomerular filtration rate (GFR) using 24-hour urine measurements. Secondary objectives included evaluation of such estimations based on age, body mass index (BMI), and pregnancy. Methods: This was a retrospective chart review of 195 patients who were ≥ 18 years of age and who had a 24-hour urine sample collected to determine GFR. Subjects were identified based on an ICD-9 search strategy for 24-hour urine sample. Demographic and laboratory data were collected from medical records and used to calculate GFR estimates. CKD-EPI, CG, MDRD-4 and generated estimates of GFR were calculated for each patient included. Results: Calculated GFR using CG, CKD-EPI, and MDRD-4 resulted in significant underestimation. Reclassification to a higher GFR proved less likely in those who were obese or elderly. As age increased, GFR decreased in both the male and female population for all creatinine based formulas except 24-hour urine collection. CG reported the highest mean average among the creatinine-based equations in all subgroups with a BMI >18.5 while CKD-EPI reported the lowest. Conclusions: While the equations evaluated did not provide an accurate measure of GFR, these methods are considered the least invasive and most convenient. Based on the results of this study, use of a 24-hour urine collection should be used when accurate estimation of GFR is warranted and measurements of inulin clearance is not feasible.