Singlet oxygen (¹O₂) is the most important cytotoxic agent in photodynamic therapy (PDT). The feasibility of using a chemiluminescence (CL) probe, 3,7-dihydro-6-[4-(2-(N′-(5-fluoresceinyl)thioureido)ethoxy)phenyl]-2-methylimidazo{1,2-a}pyrazin-3-one sodium salt (fluoresceinyl Cypridina luciferin analog, FCLA), to monitor ¹O₂ production during PDT is evaluated in vitro. Lymphoma cells were treated with various protocols of PDT. The results show that the FCLA-CL production during PDT is linearly related to the corresponding cytotoxicity, regardless of the treatment protocol. With minimum cytotoxicity and interference to the PDT treatment outcome, the FCLA-CL system is an effective means to quantify PDT ¹O₂ production and may provide an alternative real-time dosimeter.