The effects of bio-optical characteristics on lipid and biomass production rates for Emiliania huxleyi and Tetraselmis sp. were studied under different irradiances and spectra. Biomass and lipid production increased from irradiances of 50–800 μE m−2 s−1 but differed for each spectrum. The highest biomass and neutral lipid production rates for E. huxleyi occurred under a red-blue LED array, which mimicked the absorption spectra of the cell’s chlorophyll a and accessory pigments, especially fucoxanthins. Biomass and neutral lipid production for Tetraselmis sp. were enhanced under broader spectra of cool white and grow lights. Neutral lipids per cell volume were similar for the two species and inversely proportional to chlorophyll a concentrations. High biomass and neutral lipid production rates were associated with high total quantum absorption and low quantum absorption per cell. Generally, quantum efficiencies were highest for high light treatments with high total quantum absorption and high production rates. Using reconstructed quantum absorption by pigments, the bio-optical model showed that photoprotective pigments were dependent on light intensity and spectra. Quantum efficiencies increased when excluding the contribution of photoprotective pigments to total quantum absorption since they did not directly contribute to photosynthesis, but did promote higher production rates.