The volume of body fluids and electrolyte concentration is normally maintained in a very narrow range despite large fluctuations in food intake, metabolic activities and environmental stresses. Differences in hydrostatic pressures on both sides of the cell membrane are negligible, and hydration of cells depends on the osmotic difference between the intracellular and extracellular fluid. The plasma osmolality is the most important parameter for evaluating the inner water balance. The osmometer is used for measurement of plasma osmolality, but not all laboratories have one so they use different calculation methods. There are various methods for determining the plasma osmolality: direct measurement of osmolality or calculation by the following methods: Method A mOsmol/kg H2O = 2 x ([Na+] + [K+]) + [glucose] + [urea] Method B mOsmol/kg H2O = 1.86 x [Na+] + [glucose] + [urea] + 9 The purpose of this research was to determine the osmolality in three different ways, compare the obtained values with one another, say how the results are consistent and whether the methods can be compared and replaced with each other. The study included a group of 411 patients that was balanced by gender. Samples were collected at various departments of Hospital Center Sestre milosrdnice in the morning. The osmolality was measured by osmometer, in serum samples, lowering the freezing point of the solution and then calculated according to Method A and Method B with the variables measured in plasma (concentration of sodium, potassium, glucose and urea). Data analysis showed that the methods are significantly different and are not mutually comparable (P <0.0001). The individual variables showed weaker, but statistically significant correlation with the measured osmolality except urea whose concentration is well correlated (rho = 0.649; P <0.0001) and proportionally contributes to the osmotic pressure. This result indicates that the concentrations of all four molecules as four independent variables are interconnected contributing to the osmolality in a common mathematical relation. That is also demonstrated with the multiple regression model.