A reverse phase high-performance liquid chromatography (HPLC) method was developed for the simultaneous quantification of kahweol and cafestol in tissues of fresh fruits, leaves, and roasted coffee beans. The best resolution was obtained with isocratic elution of acetonitrile/water (55/45% v/v) and UV detection. A single sample preparation method carried out by direct saponification and extraction with organic solvent was standardized for all matrices. Good recovery (average of 99% for kahweol and 94% for cafestol), repeatability, and linearity were obtained. Detection limits of 2.3 and 3.0 mg/100 g were observed for kahweol and cafestol. The HPLC method was effective in quantifying these diterpenes in the different coffee matrices. The endosperm and perisperm of Coffea arabica cv. IAPAR 59 showed elevated amounts of kahweol as compared to the pericarp and leaves. On the other hand, cafestol was detected in all samples except in leaves from Coffea canephora cv. Apoatā.