There is a need to develop a questionnaire as a research tool for the evaluation of water balance in the general population. The water balance questionnaire (WBQ) was designed to evaluate water intake from fluid and solid foods and drinking water, and water loss from urine, faeces and sweat at sedentary conditions and physical activity. For validation purposes, the WBQ was administrated in 40 apparently healthy participants aged 22-57 years (37.5% males). Hydration indices in urine (24 h volume, osmolality, specific gravity, pH, colour) were measured through established procedures. Furthermore, the questionnaire was administered twice to 175 subjects to evaluate its reliability. Kendall's τ-b and the Bland and Altman method were used to assess the questionnaire's validity and reliability. The proposed WBQ to assess water balance in healthy individuals was found to be valid and reliable, and it could thus be a useful tool in future projects that aim to evaluate water balance.