Guaiazulene is one of the natural azulene derivatives, which has anti-inflammatory as well as photosensitizer property and can be activated by highly penetrating red light. The proper singlet oxygen from this activation will lead to a useful strategy for specific treating unwanted cells. Objective: To measure in vitro amount of singlet oxygen produced from PDT reaction using various guaiazulene concentrations and red laser energies. Methods: Guaiazulene were prepared by diluting in methanol and mixed with 2,2,5,5-tetramethyl-3-pyrroline-3-carboxamide (TPC) to obtain 1, 2, 5, 15, 25, 35, 100, and 500 μM. Erythrosine (10 μM in each light energies) were used as positive control. Then, irradiations were performed with various red laser energies (625 nm, 151.5 mW/cm2, distance from laser tip to bottom of the well = 8 mm, and continuous mode) at 0, 4, and 8 J/cm2 in a black 96-well plate. The amount of singlet oxygen was measured in triplicate by electron spin resonance spectrometer (Bruker, Germany) at 0, 3, and 5 minutes after irradiation. Kruskal-Wallis and Mann-Whitney U test were used for statistical analyses with significant differences at p-value < 0.05. Results: The average singlet oxygen of 5 μM group significantly different from no guiazulene group (p < 0.01). Regarding the energy, both 4 and 8 J/cm2 resulted in relatively equal amounts of singlet oxygen. Conclusion: Five μM of guaiazulene activated by 625 nm laser at energy densities 4 - 8 J/cm2 dramatically generated singlet oxygen in current in vitro setting.