This study aimed to identify and determine the carotenoids from green microalga, Scenedesmus protuberans using analytical techniques. Identification of carotenoids was realized by comparing their absorption and mass spectral data with those of reference standards available and reported values. Chromatographic data were then combined with the spectroscopic information. The separation of carotenoids was achieved by C-30 column and high-performance liquid chromatography-diode array detection was used for their determination. In the present work, the carotenoid content of S. protuberans was found to be 1.45 mg/g of violaxanthin, 2.47 mg/g of all-trans-lutein, 0.15 mg/g of all-trans-alpha-carotene, 0.55 mg/g of all-trans-beta-carotene, and 0.20 mg/g of 9 or 9-cis-beta-carotene. Due to lack of their standards, the amount of all-trans-loroxanthin and cis-isomers of other carotenoids could not be quantified. In order to validate the method, Certified Reference Material (BCR 485-Mixed vegetables) was used. In conclusion, this study can serve as a reference for the analysis of carotenoids in other microalgae.