Human milk is considered as a complete food since it constitutes all the major essential nutrients, minerals, and vitamins to sustain the life. Human milk is responsible for the evolution of an efficient immune system and organ development as well as acts as a protective barrier against inflammation and infection. In addition, it is known to be a good source for polyunsaturated fatty acids (PUFAs) which are indispensable for brain and retinal development as well as growth of a child. The amount of PUFAs in the human milk varies depending on several factors such as lactation stage, maternal diet, external environment, and maternal health. In this work, we demonstrate the optimization of a detection method for estimating PUFAs in the human milk using a UV–Visible spectrophotometer. For this purpose, fatty acids were extracted from the human milk using three different types of methods, namely Hara and Radin method (HR), Bligh and Dyer (BD) method, modified Bligh and Dyer (MBD) method. Further, we compared the efficacy of all three methods on the basis of the yield of fatty acids obtained and the solvents used for extraction. The outcomes indicated that modified BD method could be the best choice amongst the three methods tested for fatty acid extraction from the human milk. In addition, the study exploited docosahexaenoic acid (DHA) in its pure form as a standard candidate of PUFA and revealed its absorption spectrum using a spectrophotometer. A sharp absorbance peak at a wavelength of 205 nm was observed to be characteristic of DHA. Also, the height of the absorbance peak was found to be varying depending upon the concentration of DHA; revealing the linear relationship between absorbance and concentration. Therefore, we characterized the absorbance plot of fatty acids extracted from the various samples of human milk at 205 nm and noticed a trend similar and well correlated with that of DHA. Thus, we anticipate that the optimized method could be utilized as a preliminary routine test to examine the PUFA content in the human milk. Keywords: Docosahexaenoic acid (DHA), Fatty acids, Human milk, Health, UV–Visible spectrophotometer