Formation of superoxides and oxides such as dimethylselenoxide (DMSeO) from oxidative reactions of methanselenol (CH3SeH) and dimethylselenide [(CH3)2Se] is one of the important mechanisms causing Se toxicity in fish and waterfowl. Because of a lack of analytical methods for directly measuring the superoxides and oxides, the mechanisms responsible for causing deformity of fish and waterfowl have not been studied in detail. In this study, an HPLC-HG-AAS system has been used to directly measure dimethylselenoxide (DMSeO) in solution. An HPLC was used to separate DMSeO with other HG-active Se compounds such as selenomethionine (Semet) and selenite [Se(IV)], and an optimum concentration of NaHB4 and HCl was used in the HG system to maximally increase DMSeO signal and decrease interferences from other HG-active Se species, and AAS was used to determine it. A detection limit of DMSeO was <5 µg/L. Recovery of spiked DMSeO in drainage water was close to 100%. This new method will be helpful to study the mechanisms of Se toxicity in fish and waterfowl.