In this study, accelerated solvent extraction (ASE) was applied to the quantitative extraction of bixin. The effects of cyclohexane/acetone ratios on bixin extraction yield were evaluated. Acetone was used in the process of pigment extraction and also played a major role in its analysis by UV-Vis spectrophotometry. Pure cis-bixin isolated by flash chromatography and characterized by Fourier Transform Infra Red spectrometry was used as a bixin standard for qualitative and quantitative analysis of annatto extracts which were obtained by accelerated solvents extraction. UV-Vis spectrophotometry analysis shows that the extraction using 100% cyclohexane gives the lowest bixin yield. This is different from the UV-Vis spectra generated with acetone extraction. The difference in percentage of acetone shows significant effects on bixin extraction yields. The use of cyclohexane:acetone (60%: 40%) solution at 50°C for 5min heating time results the highest total bixin extraction yield (48.00%) as compared to the other solvent ratios, and to the usual extraction methods using the same solvent (29.14%). High pressure liquid chromatography analysis shows that bixin extracted by this method has purity degree of 68.16%.