Dynamic pH junction is an online focusing method in CE based on the electrophoretic mobility difference of analytes in the sample matrix and the background electrolyte. An advantage of this method over the conventional CE is that the sensitivity can be significantly improved. By injecting a long sample plug in the capillary and focusing the analytes at the pH boundary between the background electrolyte and sample matrix, the LOD can be improved by 10-100 folds. The dynamic pH junction method can be easily coupled with ESI-MS. In this work, we used this method for the analysis of microcystins (MCs). The detection limits and dynamic ranges were studied. The separation was optimized by adjusting the injection time, and concentrations and pH values of the background electrolyte. The optimization of analyte focusing leads to enhanced detection response compared to conventional injections, achieving 200-400 fold higher averaged peak heights for four microcystin (MC) variants. More importantly, this method was successfully used for the quantitative analysis of microcystins (MCs) in crude algae samples from natural water bodies, making it promising for practical applications.