Quinones are widely distributed in nature and have various bioactivities. Besides, quinones are also considered as toxicological intermediates which cause severe dangerous effects. Hereby, a sensitive, simple, and rapid method is reported for quinones determination. The proposed method employed time resolved fluorescence (TRF) microplate reader based chemiluminescent (CL) detection for the first time as a novel approach for measurement. Under pulse photo-irradiation, the unique photochemical characteristic of quinones is exploited to liberate reactive oxygen species (ROS) which reacted with photosensitized CL reagent. L-012, luminol analogue, was selected for its high sensitivity. Under our investigation, para-quinones showed high CL response when compared to ortho-quinones. A linear response was obtained for studied quinone concentrations in the range of 0.05-50 μM for 1,4-naphthquinone and of 0.05-150 μM for 2-methyl-1,4-naphthoquinone (menadione) and 9,10-anthraquinone with detection limit (blank + 3SD) of 0.01 μM. The proposed method allowed the rapid determination of large number of samples in very short time (96 sample/125 s). The proposed method was successfully applied for determination of menadione in spiked human serum.