Objective: To observe the effect of delayed serum separation on various chemistry analytes. Study Design: Quasi Experimental study. Setting: Clinical Chemistry Laboratory, Sheikh Zayed Medical College & Hospital, Rahim Yar Khan. Period: 1st December 2021 to 31st January 2022. Material & Methods: Fifty healthy volunteers of age 25-55 years from both genders were included in the study using consecutive sampling technique. Informed verbal consent was taken from all the study subjects. 4 cc blood was drawn from each subject and was divided into 3 plain tubes. 1 tube was analyzed at 1 hour for glucose, urea, creatinine, ALT, AST, ALP, sodium, potassium, chloride and calcium. 2nd tube was analyzed for same analytes 2 hours after sample collection and 3rd tube was analyzed at 4 hours. Results were recorded on a predesigned performa. Data was entered and analyzed using SPSS Software 23 for Windows. Data was presented in terms of mean and SD. P value p < 0.05 was considered significant. Results: Glucose, ALT, sodium, potassium and chloride showed statistically significant variation over time while others remained stable for up to 4 hours. There was decrease in glucose (p=0.000) while increase was observed in sodium (p=0.0001), potassium (p=0.0001), chloride (p=0.0001) and ALT (p=0.002). Conclusion: Blood samples should be transported to laboratory immediately and when received in laboratory samples should be centrifuged and processed within 2 hours as delayed transportation and separation affects many chemistry analytes and may lead to erroneous test results.