Photodynamic therapy (PDT) is a treatment modality that destroys the tumor. It activates the photosensitizer with the light of a specific wavelength, where the light is well absorbed by the photosensitizer, thus causing a fatal injury and thereby leading to a tumor necrosis. To date, a hematoporphyrin-derived photosensitizer has been widely used. It is disadvantageous, however, in that it causes a long-term photo-toxicity and has a poor selectivity for the tumor. This had led to the development of a chlorophyll-derived photosensitizer. We conducted this study to elucidate the mechanisms by which the activity of ROS is involved in the PDT using a novel type of chlorophyll-derived photosensitizer, 9-hydroxypheophorbide-α (9-HpbD-α), for the HeLa cell lines. Besides, we also attempted to determine which reaction plays a predominant role in the synthesis of ROS, either Type I reaction or Type II one, when both reactions are involved in the synthesis of ROS during the PDT using 9-HpbD-α. Our results showed not only that the activity of ROS is involved in the PDT using 9-HpbD-α in human uterine cervical cancer cell lines but also that the mechanisms of PDT are based on Type II reaction where the singlet oxygen is involved.