The separation and identification of colourants from aqueous matrices could potentially benefit the coloration industry. In this work, we report a new method that combines hydrophilic interaction liquid chromatography (HILIC) and high-resolution mass spectrometry (HRMS) for reactive dye separation and identification without employing ion-pairing agents. The conditions outlined allowed the successful separation of a mixture of four commercial reactive dyes in an aqueous solution, which consisted of CI Reactive Black 5, CI Reactive Orange 35, CI Reactive Blue 49 and CI Reactive Red 31. To further demonstrate the feasibility of this new method, we conducted deeper research into the analysis of CI Reactive Red 31 and its hydrolysis products. Based on the high efficiency of HILIC for polar compounds, and its combination with HRMS, we were able to identify several isomers of CI Reactive Red 31 and its derivatives, which were further characterised by tandem mass spectrometry. This method could potentially benefit chemical evaluations in dye applications, including synthetic processes, because it provides reliable results and simplified operation conditions compared with common traditional high-performance liquid chromatography methods.