Status of diabetes of an individual is majorly evaluated by the frequent monitoring of glucose estimation. Use of serum samples and inappropriate plasma for estimating glucose is an existing practise in Indian standard of laboratories. There is a strong evidence for occurrence of in vitro glycolysis on the above mentioned specimens. The aim is to study the pre-analytical variations on the glucose estimation of using sodium fluoride-disodium EDTA (NaF-Na2EDTA) plasma (glycolysis inhibiting anticoagulant) and determine the fact behind the activity of glycolysis inhibition on the same. Healthy volunteers 20-35 years of both genders consisting of 40 members were selected for the study, and after getting the informed consent form, random blood samples were collected to study the errors of pre-analytical i.e., mixing of NaF-Na2EDTA tube by phlebotomist (no of inversion). Difference in duration from blood collection to centrifugation and a variable in time were taken from centrifugation to analyzing the plasma sample. Comparative studies on EDTA plasma and serum sample were also carried out. The usage of the evacuated blood collection system on NaF-Na2EDTA was shown to have the complete glycolysis inhibitor among all pre-analytical errors, whereas other tubes shown considerable increase in glycolysis. Recently the use of glycolysis inhibitor tubes are come into practise only in accredited or certified laboratories. Hence the authentication of glycolysis inhibition study is mandatory for the pre-analytical variations on the same.