In order to develop a practical approach for fast and non-destructive assay of total fatty acid (TFA) and pigments in the biomass of the marine microalga Nannochloropsis sp. changes in TFA, chlorophyll, and carotenoid contents were monitored in parallel with the cell suspension absorbance. The experiments were conducted with the cultures grown under normal (complete nutrient f/2 medium at 75 μmol PAR photons/(m(2) s)) or stressful (nitrogen-lacking media at 350 μmol PAR photons/(m(2) s)) conditions. The reliable measurement of the cell suspension absorbance using a spectrophotometer without integrating sphere was achieved by deposition of cells on glass-fiber filters in the chlorophyll content range of 3-13 mg/L. Under stressful conditions, a 30-50% decline in biomass and chlorophyll, retention of carotenoids and a build-up of TFA (15-45 % of dry weight) were recorded. Spectral regions sensitive to widely ranging changes in carotenoid-to-chlorophyll ratio and correlated changes of TFA content were revealed. Employing the tight inter-correlation of stress-induced changes in lipid metabolism and rearrangement of the pigment apparatus, the spectral indices were constructed for non-destructive assessment of carotenoid-to-chlorophyll ratio (range 0.3-0.6; root mean square error (RMSE) = 0.03; r (2) = 0.93) as well as TFA content of Nannochloropsis sp. biomass (range 5.0-45%; RMSE = 3.23 %; r (2) = 0.89) in the broad band 400-550 nm normalized to that in chlorophyll absorption band (centered at 678 nm). The findings are discussed in the context of real-time monitoring of the TFA accumulation by Nannochloropsis cultures under stressful conditions.