Photodynamic therapy (PDT) has been established as a local treatment modality for several kinds of malignancies in various organs.1–7 PDT is based on the use of a light sensitive drug, a photosensitizer, which is locally applied or systemically administered. The photosensitizer meta-tetra(hydroxyphenyl)chlorin (mTHPC or Temoporfin) is one of the most potent clinically used photosensitizers to date.8–10 Its development, study and clinical use was recently summarized in a comprehensive review.11 The formulation of mTHPC in ethanol and propylene glycol, Foscan®, is in use for both curative and palliative treatment of head and neck squamous cell carcinoma (SCC).7,12 The treatment involves excitation of the administered photosensitizer with non-thermal light at the tumor site, which leads to the formation of cytotoxic reactive oxygen species.9,13–17 The amount of reactive oxygen species formed depends on the type of photosensitizer, its concentration, tissue oxygenation, and the rate of irradiation. In head & neck tumors, treatment is typically performed using a mTHPC dose of and light fluence of at a fluence rate of delivered at 652 nm.11 However, despite the fixed light fluence and administered drug dose differences in PDT response may occur. Monitoring PDT parameters such as oxygenation, light fluence, and photosensitizer concentration during therapy could provide insight in the complex and dynamic interactions that occur during PDT and could give information on the deposited PDT dose.18 Our group recently developed fluorescence differential path-length spectroscopy (FDPS) as a tool to quantify photosensitizer concentration and micro vascular oxygen saturation, a surrogate marker of tissue oxygen concentration.19,20 In previous research, we were able to show that FDPS can be used to measure photosensitizer concentration in vivo in rat liver.21 In this proof-of-concept study, our group used Foscan at as the target photosensitizer. A good linear correlation was found between the mTHPC concentration measured with FDPS and the mTHPC concentration measured by chemical extraction. As a next step towards clinical translation of FDPS for monitoring PDT in head and neck tumors, we here evaluate the performance of FDPS using a clinically relevant drug dose of in target organs for head-and-neck PDT: the lip and the tongue. From a tissue optics point of view it is more challenging to analyze oral mucosal tissues compared to liver tissue. For example, oral tissues such as the dorsum of the tongue and palate are keratinized and are effectively layered media, while the inner lip and floor of the mouth are less so. The keratinisation of the dorsal tongue is present in all mammals, although the degree of keratinisation varies among species.22 In the present study, we have investigated how accurately FDPS measures photosensitizer concentrations in these more optically heterogeneous media. Similar to our previous proof-of-concept study, chemical extraction will serve as the gold standard for mTHPC concentration in these tissues.