Determinations of fluid volumes are of importance for correct treatment of patients subjected to shock and trauma. Gas isotope ratio mass spectrometry (GIRMS) is an advanced method for analysis of stable isotopes. These can be used as tracers for measurement of various fluid volumes. In the current in vitro study, deuterium was used to determine different volumes of water simulating a range of body fluid volumes from neonates to adults. A high-precision scale gave control weights (i.e., volumes), and two methods, equilibration (EQ) and chromium reduction (CR), were compared by use of a GIRMS. The coefficient of variation was <1% when using both EQ (0.45%) and CR (0.79%). The variability was greater at small volumes, and, when regression equations for the relation between measured and calculated volumes were used as formulas, the deviation was 0.4% using EQ and 2.8% using CR at the volume of 1,000 ml. At larger volumes, the deviation when using CR approached 1%. These variations are better than previously published data using other methods. It was concluded that GIRMS is a suitable technique for fluid volume determinations in neonates as well as in adult patients, using deuterium as a tracer. EQ and CR methods were both regarded to give acceptable variabilities in this in vitro study. GIRMS may in the future increasingly be used clinically for accurate measurements of body fluid volumes.