Carapa guianensis crabwood, popularly known in Brazil as “andiroba”, is a tree that grows in South and Central America and is used by the local population mainly for its anti-inflammatory and insect anti-feeding activities. Scientific studies on this plant have led to the development of an insect-repellent candle and the investigations of the anti-inflammatory properties of its more important biomarkers—tetranortriterpenoids. These compounds, together with glycerides and fatty acids, are present in the seed oil, the most important commercial product from this plant. The growing scientific and commercial interest in “andiroba oil” has urged the development of adequate analytical methods for assessing its quality. Central composite experimental design is a useful statistical method for the development and optimization of HPLC methods, and has been used for a variety of samples. The aim of this work is to develop a HPLC method for the determination of tetranortriterpenoids in “andiroba” oil, by means of central composite experimental design, as well as to prevalidate this method.