Urine color (Ucol) as a hydration assessment tool provides practicality, ease of use, and correlates moderately to strongly with urine specific gravity (Usg) and urine osmolality (Uosm). Indicative of daily fluid turnover, along with solute and urochrome excretion, Ucol may also reflect dietary composition. Thus, the purpose of this investigation was to determine the efficacy of Ucol as a hydration status biomarker after nutritional supplementation with beetroot (880 mg), vitamin C (1000 mg), and riboflavin (200 mg). Twenty males (Mean ± SD; age, 21 ± 2 y; body mass, 82.12 ± 15.58 kg; height, 1.77 ± 0.06 m) consumed a standardized breakfast and collected all urine voids on one control day (CON), and one day after consuming a standardized breakfast and a randomized and double-blinded supplement (SUP), over 3 weeks. Participants replicated exercise and diet for one day before CON, and throughout CON and SUP. Ucol, Usg, Uosm, and urine volume were measured in all 24-h samples, and Ucol and Usg were measured in all single samples. Ucol was a significant predictor of single sample Usg after all supplements (p<0.05). Interestingly, 24-h Ucol was not a significant predictor of 24-h Usg and Uosm after riboflavin supplementation (p=0.20, p=0.21). Further, there was a significant difference between CON and SUP 24-h Ucol only after riboflavin supplementation (p<0.05). In conclusion, this investigation suggests that users of the UCC should consider riboflavin supplementation when classifying hydration status and utilize a combination of urinary biomarkers (e.g., Usg and Ucol), both acutely and over 24-h.