Colour is an important component of durum wheat quality affecting consumer’s choice. Pasta colour is the result of the carotenoid pigments accumulated in the seed endosperm and of the enzymatic activities responsible for their degradation. Thus, increasing yellow colour is a valuable goal for improving the commercial and nutritional values of durum wheat products. This work reports the set up of a micro-method for the determination of Yellow Pigment Content (YPC) in durum wheat. The novel method, although similar in principle to the Official Method AACC 14-50, allows the use of micro-amount of samples (10–100 mg), a condition that for durum wheat corresponds to a single kernel, and micro-amount of extraction solvent (water-saturated 1-butanol, 250–500 μL). These improvements together with a short extraction time lead to significant benefits from the practical and the economic point of view. The improved method has been validated using nine durum wheat cultivars extracted in 15 min with the ultrasonic bath. The YPC was calculated by reading the absorbance at 435 nm after the manual injection of an aliquot of extract directly into a UV–Vis HPLC detector. The results obtained were comparable and not significantly different from the Official AACC Method 14-50.