A novel analytical method for analysis of microbial quinones in activated sludge sample-was developed with improved reliability compared to the conventional method. The operating conditions for the extraction of quinones from activated sludge sample with a methanol-chloroform mixture and hexane were optimized. A solid phase extraction method using the Sep-Pak(R) Plus Silica, a small column packed with silica gel, was employed for the purification and separation of quinones instead of column chromatography and thin layer column chromatography used in the conventional method. The analytical operation was simplified and the analytical time was shortened by a half or two-thirds of that required for the conventional method. The recoveries of quinones were markedly increased from 30-60% with the conventional method to as high as 90-101% with the improved method, demonstrating that the reliability of the analytical results of the improved method is significantly higher than that of the conventional method. The quinone content value obtained using the improved method was twice as high as that obtained using the conventional method for the same activated sludge sample, but the quinone profiles expressed as the mole fraction of each quinone type were the same for each method.