Main objective of this study was to optimize the analytical procedures, especially extraction and saponification method for the better analysis of β-carotene in milk fat. Saponification method with suitable potassium hydroxide (KOH) concentration, use of antioxidant, saponification time, and temperature was optimized to get best extraction procedure for β-carotene . Analysis of β-carotene was carried out by using reversed phase high performance liquid chromatography (RP-HPLC) using C18 column. Chromatographic conditions best tested for the study involves methanol: tetrahydrofuran: water (MeOH: THF: H2O). The effect of time temperature combination at 45°C/30 min with saturated ascorbic acid had a pronounced effect on the β-carotene extraction from milk fat. Study also reveals that a concentration level of 10M KOH was best suited for the extraction of unsaponifiable. © All Rights Reserved.