This is the first study to fully validate a HPLC method to be applied for the simultaneous analysis of secondary metabolites from Bixa orellana (annatto) seeds. These validation parameters included linearity, selectivity, procedure accuracy, system precision, recovery and stability. For the target compounds, linear regression analyses were performed by using external calibration curves. The parameters for the calibration curves (slope, intercept, relative standard deviation of slope, relative standard deviation of intercept and correlation coefficient) are shown in Table 3.