Isolation of good quality ribonucleic acid (RNA) from mammary glands of elite animals is often hindered by invasive method of mammary tissue sampling, ethical permission, time consuming, and repeated biopsies from the same animal at different time points. The aim of this study was to optimize a protocol for RNA isolation from milk fat globules (MFG) that is suitable for transcriptome analysis. We isolated good quality RNA from milk fat of goats and buffalo milk by combining 2 methods namely Trizol and GenElute mammalian RNA isolation kit. The concentration of RNA ranges from 385 to 3000 ng/µl (1267.5 ± 186.5 ng/µL) in 20 µL of elution volume. Our improved protocol resulted in optical density (OD) ratios of RNA close to 2.0 (OD260/280 = 2.05 ± 0.01 and OD260/230 = 1.99 ± 0.03) indicating its purity. RNA integrity number (RIN) value of representative sample was 8.1 indicating suitability of RNA samples for next generation sequencing like RNA-Seq. Functional validation of total RNA isolated from MFG, were tested for the expression of milk protein genes like α-lactalbumin (LALBA), β-lactaglobulin (BLG4), β-casein (CNS2) and ribosomal protein genes like RPS23 for quantitative PCR analysis. We concluded that our results could be used to obtain high quality and abundant quantity of RNA for transcriptome analysis of mammary glands and could be serve as non-invasive method of expression analysis in other species.