In a clinical laboratory test done on daily basis to measure the concentration of a constituent in a body fluid in order to give information relevant to the clinical state of a patient. To withstand the stability of the constituent or analytes in a body fluid for a period of time when the sample is stored under various storage conditions is always meant to be challengeable. This present study has been performed on two important biochemistry analytes to study the effect of freezing and thawing on the analytes. 20 Left over serum samples obtained from the free laboratory of Biochemistry were analyzed on day 1 for baseline measurement for both the analytes followed by freezing the sample at –20˚c and thawing at room temperature. In such manner the samples were made to undergo 10 freeze and thaw cycles. The results were compared with those obtained on the first day. Mean, standard deviation, and percentage difference were evaluated statistically. The Comparison of the data showed mean change by 0.03gm/dL from day1 to day10 and for total bilirubin mean change by 0.18mg/dL from day1 to day 10 with decrease in the total bilirubin values by 4.08% and total protein by 0.76%. As a result the study concludes that total bilirubin values gradually decrease when we repeatedly freeze at – 20˚c and thaw at room temperature. Total protein is stable for 10 cycles of freezing and thawing. Hence we need to be cautious while planning the studies and research projects which take up bilirubin as the parameter where repeated freezing and thawing is required.