Fatty acid profiling has become a very useful and effective tool in the diagnosis, prevention and treatment of several diseases with cardiovascular disease being particularly important. In order to arrive at accurate conclusions that would help promote the health of individuals plagued by such diseases, not only excellent laboratory methods are required, but also very important monitoring responses to treatment. Improvements in methods of fatty acid profiling in biological systems regarding safety of extraction, precision and time for analysis are valuable. The ω-3 index (a measure of the amount of eicosapentaenoic acid, EPA, and docosahexaenoic acid, DHA, in Red Blood Cell membranes expressed as the percent of total fatty acids) is of growing interest because it has been reported to provide prognostic information regarding the risk for heart diseases. Sodium methoxide has been widely used for the determination of ω -3 fatty acids in food samples. This study demonstrates that sodium methoxide can be used effectively in RBC fatty acid profiling and determination of the ω-3 index. Briefly, the fatty acid profiles and ω-3 index of red blood cell samples were analyzed and results compared using three different methods: a two-step extraction and methylation method described by Hara and Radin, a single step extraction and methylation method described by Harris et al. and the sodium methoxide method.Our results revealed that there were no statistically significant differences (p<0.05) between the three methods for the representative fatty acids, [16:0 (p = 0.10), 18:0 (p=0.40), 18:1(ω9) (p = 0.29), 18:2(ω6) (p = 0.95), 18:3(ω3) (p = 0.50), 20:5(ω3) (p=0.56), 22:6(ω3) (p = 0.06)] and ω-3 index (p = 0.11) except for 20:4(ω6), (P = 0.02). In conclusion, we show that sodium methoxide can be used effectively in a one-step extraction and methylation procedure for high throughput analysis of fatty acids in red blood cell membranes. It is rapid (10 minute extraction and methylation), simple, safer than and as accurate as other commonly reported methods.