The study was conducted to evaluate the differences in results obtained for assays of ionized calcium (iCa +2 ) by plain and heparinised blood sample and observe for any errors in values done by ion selective electrode (ISE) method and to determine which of the collection methods could be ideal and reliable. 49 samples of heparinised and 31 plain blood samples were analyzed at lab services, Apollo Reach hospital, Karimnagar, Telangana state for iCa +2 by ISE method using radiometer analyzer and the differences in data were documented statistically by calculating the mean and SD. The results of the study showed statistically significant difference in values of iCa +2 when blood was collected in plain tube (4.7±0.2) and with heparinised collection (4.4±0.3). It appears in the study that plain tube collection for the assay is ideal.Keywords: iCa+2, calcium-neutralized lithium zinc heparin (CNLZ), heparinised and plain tube for iCa+2, iCa+2by ion selective electrode (ISE) method