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The body of literature varies significantly regarding serum and urine osmolality stability. Therefore, our aim was to investigate the stability of serum and urine osmolality at different temperatures (room temperature (RT) 4–8 °C, −20 °C) and time conditions (8 h, 24 h, 1 month). The stability study was conducted following the CRESS guidelines, including 40 serum and urine samples. Samples were aliquoted into three aliquots and stored as follows: primary tube stored at RT for 8 h; two capped aliquots stored at 4–8 °C for 8 h and 24 h; one aliquot stored at −20 °C for 1 month. To minimize imprecision error, serum and urine osmolality were measured by the freezing point depression method in triplicate on OSMOMAT 3000 (Gonotech, Germany) analyzer. Percentage difference (PD%) against baseline measurement was calculated. Deviations were assessed against a reference change value of 5.0%. The PD% for serum and urine osmolality was below 2.0% for all time/temperature conditions. For serum samples: primary tube after 8 h at RT PD% (95% CI) = 0.0% (−0.3, 0.2%); 8 h at 4–8 °C PD% (95% CI) = −0.4% (−0.7, 0.0%); 24 h at 4–8 °C PD% (95% CI) = −0.7% (−0.7, −0.6%); 1 month at −20 °C PD% (95% CI) = −2.1% (−2.4, −1.5%). For urine samples: after 8 h at RT PD% (95% CI) =0.6% (0.2, 0.9%); 8 h at 4–8 °C PD% (95% CI) = −0.2% (−0.5, 0.1%); 24 h at 4–8 °C PD% (95% CI) = −0.2% (−0.5, 0.0%); 1 month at −20 °C PD% (95% CI) = −2.0% (−3.0, −1.0%). Changes in osmolality for tested conditions for serum and urine samples, were within acceptance criteria. Reflex and add-on osmolality testing can be performed within the same day in samples kept at RT for 8 h in primary tube and within 24 h, in aliquoted refrigerated samples, without compromising the reliability of test results. For longer storage, samples should be kept at −20 °C.