We introduced a droplet-based magnetically activated cell separator (DMACS) that utilizes a hanging droplet of microliter-sized volume for dividing positive (magnetically labeled) and negative (magnetically unlabeled) cells. It enabled us to solve adhesion problem in channel and showed better separation efficiency. However, the separation efficiency was changed under variations of initial volume (the droplet volume of cell suspension before buffer injection process for dividing the hanging droplet into two fractions). Therefore, the efficiencies corresponding to initial volumes were investigated in experimental study. We found that the initial volume should be optimized to find maximum separation efficiency.