An in vivo non-invasive quantitative analysis technique was introduced for evaluating the fat composition of living marine diatoms by using Raman spectroscopy in conjunction with a chemometric method. This technique enabled the observation of real-time variations in individual lipids in diatom cells without specific treatment or fat extraction. A confocal Raman spectroscope was used to measure the marine centric diatom Thalassiosira (T.) pseudonana that was cultured under six stress conditions, and the spectral data of accumulated fatty acids were obtained. A model-based chemometrics technique, ordinary least square was then used to extract specific signals from Raman spectra obtained for a mixture of fatty acids. The levels of four major lipid moieties from diatoms were extracted simultaneously, including myristic acid, palmitic acid, palmitoleic acid, and eicosapentaenoic acid from the Raman spectra. These results indicate that Raman spectroscopy in conjunction with a chemometrics method is reliable for the quantitative determination of the lipid composition accumulated in the cells of marine diatoms.