Trade-off between growth and lipid accumulation is one of the main issues in cultivation of microalgae for biodiesel production. Advantages of two-phase cultivation systems, with growth and lipid accumulation occurring in separate tanks, have been proposed. However, because the two-phase cultivation system requires more cultivation space and complex operation procedures compared to a single tank system, its advantage for lipid production is still uncertain. Furthermore, previous studies have not explicitly determined an optimal cultivation schedule, defining timing of the transfer of cells to the second tank and harvesting in the second tank. We therefore developed a model for lipid accumulation in the microalgae Chlorella sorokiniana to compare theoretical maxima of lipid productivity in single- and two-tank cultivation systems. We first established a monitoring method of growth and lipid accumulation using a flow cytometer, and then modeled the data by a logistic equation to derive a model for lipid accumulation. This model was used to theoretically optimize cultivation methods and harvest times to maximize lipid productivity in four different cultivation systems: single- or two-tank setups, with batch or fed-batch cultures, respectively. Results theoretically demonstrated that a two-phase cultivation system is slightly more productive than a single-tank one. The mathematical model analysis indicates strategies for further improving the productivity of each cultivation system without any additional investments in cultivation systems (i.e., nutrient, light, CO2). Our model analysis approach provides a theoretical basis for determining an optimal cultivation strategy of microalgae and identifies experimental data helpful for further improvement of cultivation systems.