Evaluation of renal function should be performed as part of the follow-up during and after chemotherapy in pediatric cancer patients. The aim of this study was to compare an isotope clearance method [isotope glomerular filtration rate (iGFR)] with alternative methods to determine GFR in such patients. Isotope GFR [(99m)Tc-labeled diethylene triaminopentoacetic acid (DTPA) or (51)Cr-labeled ethylenediaminetetra-acetate (EDTA)] was measured in 36 children (112 studies) and compared with simultaneously measured creatinine clearance (CrCl), serum creatinine (SCr), and cystatin C (CysC) concentrations, as well as the results of Schwartz, Counahan-Barratt, and Cockroft-Gault formulae, using general linear mixed models. Our results showed a significant association between iGFR and CysC concentrations (p < 0.001). No linear relationship was observed between CrCl and iGFR (p = 0.7). As expected, the results of height-based formulae (Counahan-Barratt and Schwartz) had significantly (p = 0.004) better correlation to iGFR than the results of a formula based on weight (Cockroft-Gault) (p = 0.19). Despite significant linear correlation, intraclass correlation coefficients showed poor agreement. Tests of similarity between iGFR estimates showed differences between average values of GFR. Therefore, determination of iGFR remains the method of choice in estimation of GFR in cancer patients. In our study population, assay of serum CysC was the most reliable alternative method to measure glomerular function.