To determine the concentration of ionized calcium (iCa) collected in lithium heparin and gel tubes and to correlate the measured iCa with calculated iCa. Anaerobic fasting blood samples were simultaneously collected from healthy laboratory workers in lithium-heparin and gel tubes. iCa, pH, total calcium (CaT), total protein and albumin were measured. Ionized Ca was calculated with albumin and globulin values using an appropriate formula. Mean iCa in gel tubes showed a positive constant bias of 0.08 mmol/L (p < 0.001) when compared to lithium heparin results. The mean pH of blood taken in lithium heparin and gel tubes was non-significant (p = 0.3). Measured iCa poorly correlated with CaT (r = 0.2, p = 0.1) and calculated iCa (r = 0.2, p = 0.2). To evaluate the calcium status direct measurement of iCa must be done rather than using the formulae for iCa determination. In addition serum is recommended as the choice of sample for iCa determination in comparison to plasma samples.