Photodynamic therapy (PDT) is a treatment that combines the use of non-toxic dyes or photosensitizers (PS) with harmless visible light. This review series discusses the mechanisms underlying PDT, focusing on the recent advancements in the discovery and synthesis of new PS, their photochemistry, photophysics, and subcellular localization. The selection of an ideal PS for a specific application is crucial, and guidelines are provided based on factors such as toxicity, light absorption, and tissue penetration. The photochemistry of PS involves two main pathways: Type I, which involves the generation of radicals and reactive oxygen species (ROS), and Type II, which involves the formation of singlet oxygen. Understanding how light travels through tissues and the effects of absorption and scattering is essential for optimizing treatment planning and ensuring sufficient light reaches the diseased tissue. The subcellular localization of PS is a key factor in determining the effectiveness of PDT, with PS localizing in various organelles such as mitochondria, lysosomes, endoplasmic reticulum, Golgi apparatus, and plasma membranes. The use of 5-aminolevulinic acid (ALA) as a precursor for PS synthesis in tumors or cells is also discussed, highlighting its potential in improving PDT efficacy. The review emphasizes the importance of rational mechanistic studies to predict the efficacy and optimal clinical application of different PS based on their chemical structure and simple spectroscopic measurements.Photodynamic therapy (PDT) is a treatment that combines the use of non-toxic dyes or photosensitizers (PS) with harmless visible light. This review series discusses the mechanisms underlying PDT, focusing on the recent advancements in the discovery and synthesis of new PS, their photochemistry, photophysics, and subcellular localization. The selection of an ideal PS for a specific application is crucial, and guidelines are provided based on factors such as toxicity, light absorption, and tissue penetration. The photochemistry of PS involves two main pathways: Type I, which involves the generation of radicals and reactive oxygen species (ROS), and Type II, which involves the formation of singlet oxygen. Understanding how light travels through tissues and the effects of absorption and scattering is essential for optimizing treatment planning and ensuring sufficient light reaches the diseased tissue. The subcellular localization of PS is a key factor in determining the effectiveness of PDT, with PS localizing in various organelles such as mitochondria, lysosomes, endoplasmic reticulum, Golgi apparatus, and plasma membranes. The use of 5-aminolevulinic acid (ALA) as a precursor for PS synthesis in tumors or cells is also discussed, highlighting its potential in improving PDT efficacy. The review emphasizes the importance of rational mechanistic studies to predict the efficacy and optimal clinical application of different PS based on their chemical structure and simple spectroscopic measurements.