Evaluation of the stability of serum and plasma analytes under different temperatures and time points is vital since it significantly affects the pre-analytical errors. The aim of the study was to determine the stability of serum total cholesterol and triglyceride and plasma glucose of healthy individuals under different storage conditions (25-30°C and 2-8°C) and at different time points (1-hour, 4-hour, 6-hour, and 24-hour). Venous blood samples were collected from healthy individuals aged between 20-50 years after a 12-hour fast. Plasma and serum were separated and stored at different temperatures (25-30°C and 2-8°C) and different time points (1-hour, 4-hour, 6-hour, and 24-hour). Results indicated a significant difference between the mean plasma glucose concentration at room temperature (25-30°C) and refrigerated temperature (2-8°C) at 1-hour (0.072%, p = 0.05). However, there were no significant differences in mean serum total cholesterol concentrations (p = 0.633) and mean serum triglyceride concentrations (p = 0.246) between these temperatures at 1-hour time point. Regarding time points (1-hour, 4-hour, 6-hour, and 24-hour), significant differences in plasma glucose concentrations occurred at 24-hour (10.03%, p = 0.001) at room temperature (25-30°C) and 6-hour (8.79%, p = 0.003) at refrigerated temperature (2-8°C) compared to the 1-hour reference values. Additionally, there were significant differences in serum total cholesterol concentration at 4-hour (12.55%, p = 0.000) and serum triglyceride at 24-hour (29.18%, p = 0.001) compared to the reference values obtained at 1-hour at room temperature (25-30°C). No significant differences were observed among the time points (1-hour, 4-hour, 6-hour, and 24-hour) for serum total cholesterol (p = 0.507) and serum triglyceride values (p = 0.761) at refrigerated temperature (2-8°C). Plasma glucose is unstable at both room temperature (25-30°C) and refrigeration (2-8°C), with recommended testing within 6 hours at room temperature (25-30°C) and stored in a refrigerator (2-8°C) if delayed. It is better to estimate serum total cholesterol within 1-hour of collection at room temperature (25-30°C) while serum triglyceride concentration within 6 hours at the same temperature to obtain accurate and reliable results.