To determine the variation in serum Ionized Calcium values between serum separated at 0 hour and samples when serum was separated after 4 hours with clot contact. Another comparison is between sample separated immediately and serum sample stored after separation for 24 hours at 2-8 degree refrigerated temperature. This study was done to determine the pre-analytical variation due to different storage and sample segregation methods employed by different hospitals depending upon their infrastructure and human resource. This study further emphasizes that since the variation found was minimal and non-significant in serum samples, it should be the sample of choice for the measurement if iCa. The method employed for estimation was direct ISE only and no comparison is done between the methodologies.