Introduction: In outpatient settings, it is challenging to exclude acute kidney disease (AKD) based on a single serum creatinine (SCr) measurement. This retrospective study aimed to explore the usefulness of urinary biomarkers and a novel functional biomarker, a spot urine creatinine-to-osmolality ratio (sUCr/Osm), for inferring the absence of AKD. Methods: The cohort was from the ASSESS-AKI Study. ‘No AKD’ was defined as the absence of a SCr increase ≥ 26.5 μmol/L between the preceding visit and the index visit, with a three-month interval. Urinary neutrophil gelatinase-associated lipocalin (UNGAL) was selected as the representative biomarker out of six known candidates. UNGAL levels < 100 ng/mL indicated a positive test. sUCr/Osm values ≥ 7.07 indicated a positive test. Results: Of the 1,570 participants, 38.0% were female. The mean age (mean ± SD) was 64.6 ± 13.0 years, and the mean SCr level was 102.5 ± 51.4 μmol/L. The area under the receiver operating characteristic curve for UNGAL in identifying ‘No AKD’ for all participants was 0.548 (95% confidence interval: 0.495–0.600), whereas that for sUCr/Osm was 0.578 (0.525–0.630). The sensitivity of UNGAL was 0.867 (0.849–0.884), with a positive predictive value of 0.917 (0.902–0.932) and an accuracy of 0.808 (0.788–0.827). The corresponding values of sUCr/Osm were 0.926 (0.912–0.939), 0.906 (0.891–0.921), and 0.845 (0.827–0.863). In individuals whose SCr-derived estimated glomerular filtration rate was < 60 mL/min/1.73 m2, sUCr/Osm performed comparably to UNGAL. Conclusion: Using sUCr/Osm to infer the absence of AKD in outpatients with a single SCr measurement may be as effective as using UNGAL.