This study explores the influence of alkaline pH and light intensity on the performance of Neochloris oleoabundans in two-stage batch cultivation: a first stage for nitrogen-sufficient growth followed by a second stage for lipid accumulation under nitrogen starvation. The highest TAG yield on absorbed light was obtained at low light conditions when pre-cultivation occurred at pH 8 and lipid accumulation was induced at pH 10. However, a higher alkaline pH by itself appears not to enhance the starvation-induced increase in lipid contents, except when combined with high light and pre-cultivation occurs at those same conditions. Such strategy however also results in low biomass and TAG yields on absorbed light. Fatty acid composition analysis revealed that the relative fatty acid contents of the TAG pool are nevertheless independent from the light intensity and pH applied at either cultivation stage, suggesting a high specificity of N. oleoabundans cell machinery towards TAG production.