Leukotrienes (LTs) are inflammatory molecules which play an important role in triggering inflammatory diseases such as asthma, neurological diseases and diabetes. As a result, it is important to better understand their role and function in terms of disease progression and their potential value as biomarkers. Due to difficulty of isolation and quantification of these molecules from biological tissues, two different approaches were explored and compared in this study. In both methods, mouse brain tissue homogenates, prepared using the Precellys® Evolution, were used for isolation and quantification of LTs. The results showed that immunoaffinity (IA) enrichment method performed better and proved to be a better sample preparation method for simultaneous measurements of LTs ( LTB(4), LTC(4), LTD(4) and LTE(4)) when compared to the commonly used solid phase extraction (SPE) approach.