SignificanceSinglet oxygen (1O2), a type of reactive oxygen species, destroys cancer cells during photodynamic therapy (PDT). Inter-patient variations in tissue oxygen levels, dose of photosensitizer (PS), and fluence of light can affect 1O2 generation and result in suboptimal treatment efficacy in the clinic. ApproachHere, we describe a high throughput spectrometer system based on computational spectroscopy for real-time measurement of PS levels and 1O2 luminescence during PDT. The dosimeter provides a direct real-time measurement of 1O2 production in continuous-wave mode. Data were obtained from in-vitro protoporphyrin IX (PpIX) phantoms, and from in-vivo studies in mice with actinic keratosis (AK, an early form of squamous cell carcinoma) using the dosimeter. ResultsData indicated a correlation between 1O2 levels and treatment outcome (lesion shrinkage/clearance). ConclusionsThis study provides additional evidence for a correlation between 1O2 generation and AK treatment response, suggesting that 1O2 dosimetry could have utility in the dermatology clinic.