Microalgae can accumulate large proportions of their dry cell weight as storage lipids when grown under appropriate nutrient limiting conditions. While a high ratio of carbon to nitrogen is often cited as the primary mode of triggering lipid accumulation in microalgae, fast optimization strategies to increase lipid production for mixotrophic cultivation have been difficult to developed due to the low cell densities of algal cultures, and consequently the limited amount of biomass available for compositional analysis. Response surface methodologies provide a power tool for assessing complex relationships such as the interaction between the carbon source and nitrogen source. A 15 run Box-Behnken design performed in shaker flasks was effective in studying the effect of carbon, nitrogen, and magnesium on the growth rate, maximum cell density, lipid accumulation rate, and glucose consumption rate. Using end-point dry cell weight and total lipid content as assessed by direct transesterification to FAME, numerical optimization resulted in a significant increase in lipid content from 18.5 ± 0.76% to 37.6 ± 0.12% and a cell density of 5.3 ± 0.1 g/L to 6.1 ± 0.1 g/L between the centre point of the design and the optimized culture conditions. The presented optimization process required less than 2 weeks to complete, was simple, and resulted in an overall lipid productivity of 383 mg/L·d.