Studies on the effects of drying on buffalo milk are uncommon, particularly on cholesterol and their oxides (7-Ketocholesterol, 7 Keto and 25-hydroxycholesterol, 25-OH). This gap led us to this study. In triplicate, 10 L of buffalo milk were dried on spray-dryer and samples (200g) were stored for: 0, 21, 42, 63 and 84 days (n = 45). This study was proposed to validate the methodology for the cholesterol and their oxides content in buffalo milk powder and carried out by validated by analytical parameters. We did not observe any elution of interfering substance in the retention times of the cholesterol, 7-Keto and 25-OH, confirming the selectivity. The results demonstrated strong correlation among cholesterol and their oxide concentrations, demonstrated by the linearity (R2 = 0.99%). The 7-Keto’s application range was unsatisfactory, being below the lower limit (70%). The inter-day and intra-day precision for cholesterol concentrations, 7-keto and 25-OH were 2.7-7.8% and 4.0-5.6%; 3.3-6.1% and 4.9-9.3%; 2.4-5.5% and 2.7-8.8%, respectively. The proposed method for the cholesterol and its oxides detection and quantification in the buffalo milk powder was effective.