Microalgae biotechnologies are rapidly developing into new commercial settings. Several high value products already exist on the market, and systems development is focused on cost reduction to open up future economic opportunities for food, fuel and freshwater production. Light is a key environmental driver for photosynthesis and optimising light capture is therefore critical for low cost, high efficiency systems. Here a novel high-throughput screen that simulates fluctuating light regimes in mass cultures is presented. The data was used to model photosynthetic efficiency (PEµ, mol photon-1 m2) and chlorophyll fluorescence of two green algae, Chlamydomonas reinhardtii and Chlorella sp. Response surface methodology defined the effect of three key variables: density factor (Df, 'culture density'), cycle time (tc, 'mixing rate'), and maximum incident irradiance (Imax). Both species exhibited a large rise in PEµ with decreasing Imax and a minimal effect of tc (between 3-20 s). However, the optimal Df of 0.4 for Chlamydomonas and 0.8 for Chlorella suggested strong preferences for dilute and dense cultures respectively. Chlorella had a two-fold higher optimised PEµ than Chlamydomonas, despite its higher light sensitivity. These results demonstrate species-specific light preferences within the green algae clade. Our high-throughput screen enables rapid strain selection and process optimisation.