The carotenoid content of microalgae is an important quality parameter for nutritional and biological value. Determination of carotenoid content by spectroscopic and chromatographic methods is expensive and time-consuming. This study deals with the determination of carotenoids (0-carotene, zeaxanthin, neoxanthin, lutein and a-carotene) in Chlamydomonas reinhardtii by chromatography combination with principal component regression (PCR) and partial least squares (PLS) methods. Although, the chromatographic peaks of zeaxanthin and lutein were not separated by high performance liquid chromatography, PCR and PLS methods were employed successfully for the prediction of carotenoids in Chlamydomonas reinhardtii. It was found that the root mean square errors of prediction (RMSEP) for the validation set (r12=0) of PLS of 0-carotene, zeaxanthin, neoxanthin, lutein and a-carotene was 0.0500, 0.4300, 0.0252, 0.2800, and 0.1320, respectively. PLS method was used to improve resolution and quantitative determination of carotenoid overlapped highly in complicated natural samples.