BACKGROUND Plasma osmolality (POsm) is a gold standard to assess hydration status but requires venipuncture. POsm obtained by lancing a digit, a source of capillary puncture blood (CAP), has not been validated. This study compared POsm from CAP versus venous blood (VEN) and validated its sensitivity to detect dehydration. METHODS Healthy young adults (Study A: n=20 men, 22 women; Study B: n=23 men, 23 women) participated. In Study A, CAP and VEN were compared under controlled euhydration meeting dietary reference intakes for water (3.7 L men, 2.7 L women). In Study B, CAP was assessed for sensitivity to detect dehydration with receiver operating characteristic analysis over two 24 h periods: euhydration for 24 h followed by water restriction over 24 h. POsm was measured using freezing point depression. RESULTS For all subjects, CAP POsm (283.0±3.9 mOsm/kg) was not significantly different (p=0.07) from VEN (284.2±3.5) during euhydration and met analytical goals for individuality and heterogeneity. When outliers (n=3) were eliminated, mean difference was -1.6 (±3.2) lower (p<0.01) with CAP. Fluid restriction increased (p<0.001) CAP POsm (284.0±4.4 to 292.8±5.2 mOsm/kg), achieving excellent accuracy (0.92) and sensitivity (89.1%) to predict mild dehydration (2% body mass loss). CONCLUSIONS POsm via CAP exhibited similar coefficients of variation and analytical goals compared to VEN combined with excellent accuracy and sensitivity to detect dehydration. Although CAP values were approximately 2 mOsm/kg lower than VEN, CAP appears an adequate substitute for tracking changes in non-clinical settings.