Nuclear medicine (NM) methods play an important role in the evaluation of renal function in a wide range of clinical indications. The aim of our study was to evaluate the correlation between measured GFR (mGFR) obtained by the three-plasma sample slope-intercept NM method (TPSM) - reference method vs. estimated GFR (eGFR) using Fleming's single plasma sample method (SPSM) at 120 min, 180 min, and 240 min and correlation of reference method with eGFR with camera-based Gates' protocol. A total of 82 subjects (33 male/49 female) with a mean age of 54.87 ± 15.65 years were included and mGFR value was obtained by the three-plasma sample slope-intercept NM method and eGFR was obtained with Fleming's single sample method. eGFR was also quantified with the camera-based Gates' protocol after i.v. application of [99mTc]Tc-DTPA. Our study revealed a very strong positive significant correlation between all three SPSMs with the TPSM as the reference method. Between the Gates' method and the TPSM in the group of patients with mGFR ≥ 61-84 mL/min/1.73 m2 and mGFR ≥ 84 mL/min/1.73 m2, a moderate positive statistically significant correlation was obtained. The SPSM method shows a very strong correlation with the reference and low bias in all three groups of patients and can be routinely used for GFR estimation.