Background: Researchers have increasingly sought noninvasive methods to determine health and nutritional status in humans. Easy and painless to collect, human urine is a source of noninvasive biomarkers. Objective: We aimed to explore the relation between systemic oxidative stress biomarkers excreted in urine and urinary osmolality (Uosm). Design: The current trial was a descriptive, cross-sectional study. We collected seventy-eight samples of 24-h urine in preschoolers who were attending daycare centers in the Western Highlands province of Quetzaltenango, Guatemala. After we measured the total urine volume (Uvol), the aliquot was stored for the later determination of Uosm as a hydration biomarker and to measure 15-isoprostane F-2t (F2-Iso) and 8-hydroxydeoxyguanosine (8-OHdG) as biomarkers of cellular oxidation with the use of ELISA assay kits in Spain. Descriptive statistics and linear [Spearman rank-order (r(s))] and non-linear (goodness-of-fit) correlations were performed. Results: Twenty-four hour Uvols ranged from 65 to 1670 mL, whereas the Uosm varied between 115 and 1102 mOsm/kg. With respect to oxidative biomarkers, the 24-h urinary output of F2-Iso and 8-OHdG had median values of 748 and 2793 ng/d, respectively. The Uvol correlated inversely and significantly with the concentrations of both oxidative biomarkers (F2-Iso r(s) = -0.603, P < 0.001; 8-OHdG r(s) = 0.433, P < 0.001), whereas the Uosm was correlated in a direct manner (F2-Iso r(s) = 0.541, P < 0.001; 8-0HdG = 0.782, P < 0.001) when analyzed as a concentration. Associations were weaker when they were analyzed as the total 24-h production. Conclusions: Preschool children from the Western Highlands of Guatemala show strong correlations between hydration status measured through the use of Uosm and biomarkers of oxidative stress in urine. Thus, a relatively superior hydration status is associated with a quantitative reduction in urinary excretion of systemic oxidation products.