Turmeric is a plant in the Zingiberaceae family which contains curcuminoids as anticancer agents and has been widely used as a main ingredient in curry powder. However, there is a lack of suitable high performance liquid chromatography-diode array detection (HPLC-DAD) methods for reducing sample preparation time and peak resolution improvement of curcuminoids (bisdemethoxycurcumin, demethoxycurcumin, and curcumin). No significant differences in yield concentrations were observed after 60 min of heat-refluxed extraction (p < 0.05). Simultaneous chromatographic separation of all three curcuminoids achieved satisfactory results with a separation factor of 1.08 and a resolution factor of 3.39 with validation results in compliance with FDA guidelines. The expanded relative measurement uncertainty results 5.71-6.60 complied with CODEX draft. The method was successfully applied to the turmeric samples (n = 107, range 2.70-4.41 g/100 g, total curcuminoids 3.58 g/100 g). These results show that heat-refluxed extraction can be carried out easily with excellent precision and accuracy of total curcuminoids in turmeric samples.