Blood acetaldehyde levels are often measured to elucidate individual differences and pharmacokinetics of alcohol metabolism due to the gene polymorphisms of aldehyde dehydrogenase (ALDH2). Blood acetaldehyde levels are of great interest in the field of addictive behaviors, particularly drunk driving behaviors. Conventional blood acetaldehyde concentration measurements are direct and derivatization analyses. Because direct analysis is problematic in that acetaldehyde disappears during storage due to its high volatility and reactivity, derivatization analysis is often performed for acetaldehyde determination in biological samples. The conventional derivatizing method using 2,4-dinitrophenylhydrazine for determination of aldehydes unfortunately produces interfering substances from ketones and carboxylic acids. Therefore, we applied the derivatization method using 9,10-phenanthrenequinone (PQ), reported recently for the interference-free determination of aldehydes. As a result of optimizing the derivatization conditions, a linear calibration relation-ship (R²=0.9994) in the range of 3-100 μM indicated strong results. The limit of detection and quantification was 1 μM and 3 μM, respectively. The intra- and inter-day precision were both below 13.0% and accuracy was in the range of −8.5% to 2.5%. The extraction recoveries of acetaldehyde in human plasma ranged from 80.4% to 106.8%. The derivative was stable at 4 ℃ for 2 days. When acetaldehyde was added in human plasma and derivatized with PQ, acetaldehyde could be quantified by liquid chromatography-mass spectrometry. However, after drinking alcohol, acetaldehyde was measured in healthy subjects with ALDH2*1/*2, but not with ALDH2*1/*1. Acetaldehyde was difficult to detect in subjects with ALDH2*1/*1, as they are rapid metabolizers of acetaldehyde resulting in low concentrations of acetaldehyde. Acetaldehyde was detected in those subjects with ALDH2*1/*2, however, the measured acetaldehyde levels varied between the analytical methods.