Objective To date, no reference interval is available for lipid profile, including total cholesterol (TC), triglycerides (TGs), high-density lipoprotein (HDL), or low-density lipoprotein (LDL)-cholesterol, etc., in a non-fasting state. Hence, the study was taken up with the objective of exploring the possibility of establishing a reference interval for non-fasting lipid profile consisting of serum TC, TG, LDL, HDL, and very low-density lipoprotein (VLDL) cholesterol. Materials and Methods A total of 1,350 apparently healthy subjects, including 636 healthy men and 714 healthy women of 18 years and beyond of age, were enrolled in the study. Reference individuals were recruited using cluster sampling method from various villages and semi-urban regions irrespective of their sex, religion, socioeconomic status, or any other demographic profile, and samples were analyzed in Beckman Coulter AU480 analyzer. Results The mean age of 1,350 participants was 38.23 ± 15.94 years. We found that all the test parameters require a different reference interval than the established fasting reference range, except for HDL cholesterol in females. The data were subdivided into subjects below 40 years, between 40 and 60 years, and older than 60 years of age. All five parameters in the lipid profile were individually analyzed and were compared age group-wise and gender-wise with the total study population. Significant differences in the various dataset were found. Conclusion A shift toward non-fasting lipid interval measurement is, thus, a piece of evidence-driven mechanism. Even from a patient's perspective, it sets in ease and convenience in lipid-profile testing, subsequently leading to a more compliant cardiovascular management and monitoring.