Background Serum osmolality levels are measured to determine acid-base and electrolyte imbalance in serum. In cases where measurement is not possible, the serum osmolality value can be calculated by various calculation methods. In this study, we compared the Worthley osmolality calculation method which is used most frequently mentioned in literature and the measurements made with vapor pressure osmometer used in our laboratory. We compared whether there was a difference between the results obtained by measurement and calculation method in different age groups. Methods 221 serum samples of patients who were admitted to the Eskisehir Osmangazi University Hospital Biochemistry Laboratory between December 2016 and May 2018 were included in this study. Glucose, blood urea nitrogen and sodium values were recorded to determine the calculated osmolality values of the patients. Results There was a statistically significant difference between the measured osmolality values and the calculated osmolality values of the patients (p < 0.001). When compared according to age groups, there was a significant difference between calculated osmolality values (p = 0.006), but there was no difference in measured osmolality values (p = 0.787) in different age groups. It has been observed that this difference in the calculated osmolality values between the age groups is derived from the adult group (18-65, p < 0.001). Conclusion Our results showed that it is not reliable to calculate serum osmolality values, especially in the adult age group. According to our results the calculated osmolality values are higher than our measured osmolality values.