Ethylene glycol is commonly found in automobile antifreeze and used as a solvent in a variety of commercial products. In Thailand, a large number of products containing ethylene glycol are manufactured and used in various industries resulting in toxicological problems in workplaces and in the environment. Occupational ethylene glycol exposure occurs via ingestion, inhalation and contact with eye and skin. Thus, the level of serum ethylene glycol and its metabolite should be monitored in order to protect worker from its toxicity. The objective of this study is to set an analytical method of serum ethylene glycol and urine glycolic acid in order to monitor occupational exposure to ethylene glycol. This study used 90 subjects categorized into three groups: control, indirect exposure, and direct exposure to ethylene glycol. The analysis of serum ethylene glycol was preformed by GC-FID. Urine glycolic acid was determined by the HPLC-UV technique. The recoveries of serum ethylene glycol and urine glycolic acid were 99.5% and 100.5%, respectively. The measured within and between run CV imprecision of serum ethylene glycol and urine glycol acid were 2.2%, 2.5%, 2.6% and 3.8% respectively. From this study it is found that the average range of serum ethylene glycol concentrate were 0.94  0.56 mg/dL and 66.95  24.10 mg/g of creatinine for urine glycolic acid in workers directly exposed to ethylene glycol. The correlation coefficient between serum ethylene glycol and urine glycolic acid was 0.860. Therefore, serum ethylene glycol and urine glycolic acid are appropriate biological indicators for monitoring occupational exposure to ethylene glycol.