Photodynamic therapy (PDT) is a viable treatment option for a wide range of applications, including oncology, dermatology, and ophthalmology. Singlet oxygen is believed to play a key role in the efficacy of PDT, and on-line monitoring of singlet oxygen during PDT could provide a methodology to establish and customize the treatment dose clinically. This work is the first report of monitoring singlet oxygen luminescence in vivo in human subjects during PDT, demonstrating the correlation of singlet oxygen levels during PDT with the post-PDT photobiological response.