Alcohol-free beer is increasingly marketed with the claim “isotonic”. According to the European Food Safety Authority (EFSA), isotonic beverages should have an osmolality in a range of 270–330 mOsmol/kg. A method to determine osmolality in beer using an automatic cryoscope was applied and validated in this study. Isotonic and hypotonic beers can be measured directly, while hypertonic beers have to be diluted into the linear range of the instrument. As proven in several different beer matrices, the assay was linear with an average correlation coefficient of 0.998. The limits of detection and quantitation were 2 and 10 mOsmol/kg, so that the sensitivity of the method was judged sufficient to control the isotonic range. The measurement uncertainty expressed as coefficient of variation was less than 1% interday. The applicability of the method was proven by measurement of 86 beer samples. Our study has shown that the cryoscopic method is fit for the purpose to validate claims of isotonicity in food control.