Photodynamic therapy (PDT) is a treatment modality that has gained rapid popularity in both research and clinical settings over the past 20 years. PDT involves harmless red/near-infrared light excitation of non-toxic photosensitizers to generate reactive molecular species (RMS) that can induce tissue damage and/or cell death. In addition, the fluorescence signal generated from the photosensitizer can also be used for optical imaging. These effects have been harnessed for image-guided treatment of cancer and other diseases. As PDT gains popularity, it is crucial to understand and monitor different factors that could impact overall treatment efficacy. These factors include, but are not limited to, the RMS yield of photosensitizers, the distribution of photosensitizers in tissue, and the PDT activation depth in tissues. Our work focused on developing methodologies and devices to characterize and improve PDT treatment. In collaboration with the FDA, we developed a cell-free assay to rapidly and more quantitatively determine the potential phototoxicity of fluorescent probes through the measurement of singlet oxygen. We also developed a method to compare the maximal PDT activation depth of FDA-approved photosensitizers (BPD and PpIX) in the brain. We found that BPD can be activated 50% deeper into brain tissues compared to PpIX at the same radiant exposure. Next, we tested the ability of a 3D imaging system, Fluorescence Laminar Optical Tomography (FLOT), to image the distribution of photosensitizers in the rodent brain. We demonstrated that FLOT could accurately map the photosensitizer distribution up to 0.5 mm in tissues. Lastly, we developed an autofluorescent-based endoscopic imaging system to measure the metabolic impact of PDT on cancer and normal tissues, finding that PDT leads to significant changes in tissue metabolism immediately after treatment. In summary, we have developed a series of systems that can aid in PDT treatment optimization in three major ways:1) rapidly quantifying the singlet oxygen production of photosensitizers, 2) more accurately measuring a photosensitizers localization and activatable depth, and 3) developing the ability to measure a tissues response to PDT in real-time.