Abstract Background An accurate and reliable measurement of glomerular filtration rate (GFR) is essential to monitor for rejection and disease progression post-kidney transplantation. Measured GFR (mGFR) is the current gold standard. However, it has scarce availability at many centers and is labor intensive. Newer estimated GFR (eGFR) methods containing cystatin-C and/or other biomarkers have not been clinically validated in a post-transplant cohort. In this study, we evaluated a new NMR-based GFR (GFRNMR) equation that contains serum creatinine, cystatin C, myoinositol, and valine in post-kidney transplant recipients in a clinical routine setting. Methods Venipuncture for serum collection was performed immediately prior to mGFR measurement with urinary iothalamate clearance in 67 post-kidney transplant recipients. Serum was stored at 4°C and measured by NMR no later than four days after collection. eGFR was assessed using the following equations: i) GFRNMR, ii) CKD-EPI2021Cr (creatinine) and iii) CKD-EPI2021Cr,Cys (creatinine and cystatin-C). Bias was calculated as “eGFR—mGFR” for all equations. The Bootstrap method was employed to assess pairwise significance levels between bias distributions interquartile ranges (IQR), and P-value correction was determined with the Benjamini & Hochberg method. Precision, which was defined as percentage of samples within ±30% and ±15% from the mGFR value (P30 and P15, respectively), was calculated for each equation. Pairwise comparisons were made using McNemar’s Chi-squared and Benjamini & Hochberg correction. Precision was defined as the proportion of eGFR values concordant with mGFR values by CKD clinical stage. Results P30 for GFRNMR was significantly higher than CKD-EPI2021 Cr,Cys (97% vs 84%, P < 0.01) and higher than CKD-EPI2021 Cr (88%, P = 0.08). Similarly, P15 for GFRNMR was 63%; higher than for CKD-EPI2021 Cr (54%, P = 0.35) and CKD-EPI2021 Cr,Cys (51%, P = 0.19). Bias IQR for GFRNMR was 9.5 mL/min/1.73 m2 (median bias 2.0 mL/min/1.73 m2); significantly smaller than for CKD-EPI2021 Cr (Bias IQR 15.55 mL/min/1.73 m2, P < 0.05; median bias 1.47 mL/min/1.73 m2), and smaller than CKD-EPI2021 Cr,Cys (Bias IQR 14.4 mL/min/1.73 m2, P = 0.09; median bias 2.3 mL/min/1.73 m2). A broad range of medications such as immune modulators, and comorbidities such as hypertension, thyroid dysfunction, and coronary artery disease, did not interfere with GFRNMR performance. Conclusion NMR-based eGFR was less biased, more precise, and more accurate compared to creatinine and cystatin-C CKD-EPI eGFR equations in a post-kidney transplant setting. These findings suggest GFRNMR may more closely resemble mGFR in post-transplant patients and warrants further study on its potential use to improve clinical management in this patient group.