Many physiological processes rely on polyunsaturated fatty acids (PUFAs), which are present in cellular membranes. Understanding the roles and consequences of PUFAs in health and illness requires accurate study of these compounds. This research compares and contrasts traditional methods with more streamlined approaches to PUFA analysis, with an emphasis on the extraction and methylation processes. By merging the extraction and methylation processes into one, we were able to streamline the traditional procedure. The novel approach has been tested on a wide range of biological materials, including human specimens, animal tissues, and cultured cells. While both methods recover long chain fatty acids from tissue samples, statistical analysis shows that the streamlined approach is far more effective than the conventional method. However, there is no change in the relative content of fatty acids between the two methods. By streamlining the process, we can save a lot of time and resources while lowering the risk of sample contamination and loss.