This study aimed on modern analytical techniques for the isolation, separation and structural identification of the essential bioactive carotenoid Lutein, from green microalga, Chlorella saline. Identification was done by comparing their absorption and mass spectral data with those of reference standard values reported. The extract is separated by selective C-18 columns and the data were then combined with spectroscopic information. Structural assignment of the separated compound is done by HR-MS. The results of the spectral investigation showed that the isolated pigment showed absorbance peak at 445 nm. Total luminescence spectra were recorded by measuring the emission spectra in the range 350 -720 nm at an excitation wavelength of 455 nm. The excitation-emission matrices were recorded and two basic fluorescence regions have been obtained. The compound was resolved within 436 min by using a C18 column with a flow rate at 1 ml/min and detection at 450 nm. The compound was detected by a High Resolution Orbitrap-MS with regard to specificity and sensitivity (with limits of detection ranging from 1.0 to 3.8 pg mu L-1). (C) 2016 Elsevier Ltd. All rights reserved.