Introduction: A growing body of evidence indicates that Homocysteine acutely rises as a side effect of exposure to Nitrous oxide during surgery in adults. Under normal conditions, Homocysteine is remethylated back to Methionine by the enzyme Methionine synthase which requires the reduced form of Vitamin B12 as a coenzyme and 5-Methyltetrahydrofolate as the methyl donor. Exposure to N2O inhealthcare workers is associated with alterations of Vitamin B12 metabolic status. Aim: To find the Effect of Chronic Exposure to Trace Anaesthetic Gases on Plasma Homocysteine levels in Operating Room Personnel. Objective of my study is to find plasma Homocysteine levels in operating room personnel compared to non exposed. Materials and Methods: This study is conducted on 60 personnel. A total of 30 subjects exposed to waste Anaesthetic gases for a minimum of 5 years in unscavenged operation theatres and also 30 controls who were not exposed to Anaesthetic gases were selected at random to compare. From all the cases and controls detailed information pertaining to various epidemiological parameters and evaluated the level of serum Homocysteine by using ADVIA Centaur and ADVIA Centaur XP systems. Results: Mean Homocysteine value in over all analysis OT exposed group is 13.285 μmol/L and in controls is 10.545 μmol/L which are not equal. The p-Value of 0.00135 is “statistically significant”, there is a difference in the Homocysteine levels between the two groups which is because of an effect of the Anaesthetic gas exposure. Conclusion: Subjects in the exposed group are distributed in the Homocysteine values of >12 μmol/L while the subjects in the control group are distributed within the Homocysteine value of <12 μmol/L. Statistical analysis showed significant difference in Homocysteine levels between OT exposed and non exposed groups. So we conclude that long term exposure to trace Anaesthetic gases like Nitrous oxide can lead to elevated Homocysteine levels in health care workers.