Freedom of (prolonged) thirst is considered to be of paramount importance for animal welfare. This emotion normally results from dehydration, which can be measured using physiological indicators. Because no reliable physiological indicator for thirst was available for broilers, we aimed to identify such a measure in this study. This indicator would ideally be integrated into quality control systems in commercial slaughter plants. In the first experiment, water deprivation was manipulated systematically by withdrawing water for different durations (total water withdrawal for 0 (control), 24, 36, or 48 h, or a 10-d period with restricted access to water for 2 times 10 min per day). A significant decrease in drained blood content and BW occurred from 36 h of total water deprivation onward (both P = 0.03), whereas long-term restricted access tended to decrease drained blood content (P = 0.05). No effect of water deprivation or restriction on skin turgor was found. In the second experiment, water was withdrawn for 0 (control), 6, 12, 24, or 48 h. Plasma chloride concentration was increased after 6 h of water withdrawal, but did not rise further with longer withdrawal. If assessed at slaughter, chloride will thus mainly reflect the catching-to-slaughter interval. In contrast, plasma creatinine and hematocrit levels showed a numerical decrease after 6 h of water withdrawal, but rose again after prolonged withdrawal. Plasma creatinine values were significantly higher in 24-h-deprived birds than in 6-h-deprived birds (P < 0.01), allowing for discernment between water withdrawal during catching and transport from dehydration that had occurred on the farm. Blood sodium concentrations and plasma osmolality showed a steady increment between 0 and 24 h of water deprivation (P < 0.001 and P < 0.001 for both), and may thus be used to assess the combined effects of water deprivation on farm and during the catching-to-slaughter interval. These findings may form the basis of an on-farm or at-slaughter test that could be included in integrated animal welfare assessment schemes.