Background Endogenous molecules that provide an unbiased and a precise evaluation of kidney function are still necessary. We explored the potential of clearance of D-serine, a rare enantiomer of serine and a biomarker of kidney function, as a measure of glomerular filtration rate (GFR).Methods This was a cross-sectional observational study of 200 living kidney transplant donors and recipients enrolled between July 2019 and December 2020 in a single Japanese center, for whom GFR was measured by clearance of inulin (C-in). Clearance of D-serine (C-DSer) was calculated based on blood and urine levels of D-serine, as measured by two-dimensional high-performance liquid chromatography. Analytical performance was assessed by calculating biases. Utilizing data from 129 participants, we developed equations for C-in based on C-DSer and C-cre using a linear regression model, and the performance was validated in 68 participants.Findings The means of C-in and C-DSer were 66.7 and 55.7 mL/min/1.73 m 2 of body surface area, respectively, in the entire cohort. C-DSer underestimated C-in with a proportional bias of 22.0% (95% confidence interval, 14.2 −29.8%) and a constant bias of -1.24 (-5.78−3.31), whereas the proportional bias was minor to that of C-cre (34.6% [31.1−38.2%] and 2.47 (-1.18−6.13) for proportional and constant bias, respectively). Combination of C-DSer and Ccre measured C-in with an equation of 0.391 £ C-DSer + 0.418 £ C-cre + 3.852, which reduced the proportional bias (6.5% [-0.2−13.1%] and -4.30 [-8.87−0.28] for proportional and constant bias, respectively). In the validation dataset, this equation performed well with median absolute residual of 3.5 [2.3−4.8], and high ratio of agreement (ratios of 30% and 15% different from C-in [P 30 and P 15 ] of 98.5 [91.4−100] and 89.7 [80.0−95.2], respectively).Interpretation The smaller proportional bias compared to that of C-cre is an advantage of C-DSer as a measure of Cin. Combinational measurement of D-serine and creatinine, two endogenous molecules, has the potential to serve as a measure of GFR with precision and minor biases and can support important clinical decisions.