Cylindrospermopsin (CYN) is an emerging freshwater cyanobacterial toxin, and its reports on toxicity toward the human liver and kidney tissues has drawn a lot of attention. An appropriate analytical method is necessary to determine the presence of this emerging cyanobacterial toxin in water resources including drinking water; therefore, it is necessary to develop a sensitive analytical method for CYN detection. In this study, we developed a simple and sensitive analytical method for CYN detection using liquid chromatography-tandem mass spectrometry (LC-MS/MS) using direct injection. The method was validated for linearity of calibration, limit of detection, limit of quantitation, accuracy, and precision. The limit of detection and quantitation were in the range of 0.029 μg/L and 0.091 μg/L, respectively. Accuracy and precision were also obtained within an acceptable range. The optimized method was used to measure the concentrations of CYN in the surface water from each weir areas of the Geum River, Nakdong River. Additionally, this method was applied to samples of drinking water obtained from the treatment plants of the Geum River, Nakdong River for each process.