Chlorin e6 is a well-known photosensitizer used in photodynamic diagnosis and therapy. A method for identifying and purifying a novel process-related impurity during the synthesis of chlorin e6 has been developed. Its structure was elucidated using NMR and HRMS. This new impurity is formed from chlorophyll b rather than chlorophyll a , which is the source of chlorin e6. The intermediates formed during chlorin e6 synthesis were monitored using HPLC-mass spectrometry. This new impurity was identified as rhodin g 7 7 1 -ethyl ester, the structure of which remains unknown to date. The cytotoxic effects of this novel compound in both dark and light conditions were studied against five cancer cell lines (HT29, MIA-PaCa-2, PANC-1, AsPC-1, and B16F10) and a normal cell line (RAW264.7) and compared to those of chlorin e6. Upon irradiation using a laser at 0.5 J/cm 2 , rhodin g 7 7 1 -ethyl ester demonstrated higher cytotoxicity (2-fold) compared to chlorin e6 in the majority of the cancer cell lines. Furthermore, this new compound exhibited higher dark cytotoxicity compared to chlorin e6. Studies on singlet oxygen generation, the accumulation in highly vascular liver tissue, and the production of reactive oxygen species in MIA-PaCa-2 cancer cells via rhodin g 7 7 1 -ethyl ester correspond to its higher cytotoxicity as a newly developed photosensitizer. Therefore, rhodin g 7 7 1 -ethyl ester could be employed as an alternative or complementary agent to chlorin e6 in the photodynamic therapy for treating cancer cells.