The effective dosimetry for photodynamic therapy (PDT) can be specified by direct measurement of singlet oxygen (1O2) production. The purpose of this study was to investigate the feasibility of a newly developed photomultiplier tube (PMT)-based singlet oxygen detection (SOD) system. The lowest and highest 1O2 concentrations detectable by the PMT-SOD system were 15nM and 10μM, respectively. Dose-dependent quenching, by NaN3, of the fluorogenic reaction was observed, which was negatively correlated with the 1O2 level measured by the PMT-SOD system. The lifetime of 1O2, as measured by the PMT-SOD system, was found to be lengthened when H2O was replaced with deuterium oxide. 1O2 photon counts were significantly and dose-dependently correlated with intracellular fluorescence intensity after photosensitizer treatments. In vitro cell viability test and in vivo xenografted-tumor mass shrinkage showed a positive association between PDT-induced cytotoxicity and 1O2 production concomitantly measured by the PMT-SOD system. It was concluded that the PMT-SOD system is capable of measuring 1O2 production directly and accurately, demonstrating that this system can be useful in the determination of dosimetry for PDT.