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The cryptophyte Rhodomonas salina is widely used as feed for copepod cultures. However, culturing conditions to obtain high-quality algae have not yet been efficiently optimized. Therefore, we aimed to develop a cultivation protocol for R. salina to optimize its nutritional value and provide technical recommendations for later large-scale production in algal photobioreactors. We studied photosynthesis, growth, pigments, fatty acid (FA) and free amino acid (FAA) composition of R. salina cultured at different irradiances (10–300 μmol photons m−2 s−1) and nutrient availability (deficiency and excess). The optimal range of irradiance for photosynthesis and growth was 60–100 μmol photons m−2 s−1. The content of chlorophylls a and c decreased with increasing irradiance while phycoerythrin peaked at irradiances of 40–100 μmol photons m−2 s−1. The total FA content was maximal at optimal irradiances for growth, especially under nutrient deficiency. However, highly unsaturated fatty acids, desired components for copepods, were higher under nutrient excess. The total FAA content was highest at limiting irradiances (10–40 μmol photons m−2 s−1) but a better composition with a higher fraction of essential amino acids was obtained at saturated irradiances (60–140 μmol photons m−2 s−1). These results demonstrate that quality and quantity of FA and FAA of R. salina can be optimized by manipulating the irradiance and nutrient conditions. We suggest that R. salina should be cultivated in a range of irradiance 60–100 μmol photons m−2 s−1 and nutrient excess to obtain algae with high production and a balanced biochemical composition as feed for copepods.