Purification and characterization of milk oligosaccharides is a challenging process due to the complexity of the constituent oligosaccharides, which behave differently under various chemical treatment procedures, and often lose their structural properties in the process. Rathi cow’s milk is widely used in the Rajasthan region of India for its nutritional and medicinal benefits. In our earlier work, we were able to show that the medicinal properties of Rathi cow’s milk are attributed to its oligosaccharide content. Here, we aim to present an optimized method for the purification and analysis of oligosaccharides present in Rathi cow’s milk. Contrary to the freeze transport methods used earlier, we treated the collected milk with ethanol for preservation, followed by microfiltration, lyophilization and fractionation on silica gel (60-120 mesh size) column chromatography coupled with chloroform/methanol-mediated gradient elution. Fractions were analyzed for sugar content via the phenol–sulfuric acid method. Fraction homogeneity was confirmed using high-performance liquid chromatography. Isolated analytes were treated with acetic anhydride/pyridine (1:1, v/v) to form less polar oligosaccharide derivatives, which could then be easily visualized and semi-quantitated using partition chromatography (thin later and paper) with chloroform/methanol. Structural identities of the purified oligosaccharides were determined using a combination of mass spectrometry and NMR (¹H, ¹³C, HSQC, TOCSY, COSY, HMBC) techniques. Our results clearly demonstrate that the ethanol based-preservation, transport and purification of oligosaccharides is a simple and robust method for the analysis of Rathi cow’s milk oligosaccharides. Furthermore, the acetylation of purified oligosaccharides allows for rapid analysis on thin chromatography, which is quite cost-effective compared to other analytical methods.