Refill liquids for electronic cigarettes are an important area of research due to the health safety and quality control of such products. A method was developed for the determination of glycerol, propylene glycol, and nicotine in refill liquids using liquid chromatography, coupled with tandem mass spectrometry (LC-MS/MS) in multiple reaction monitoring (MRM) mode with electrospray ionisation (ESI). Sample preparation was based on a simple dilute-and-shoot approach, with recoveries ranging from 96 to 112% with coefficients of variation < 6.4%. Linearity, limits of detection and quantification (LOD, LOQ), repeatability, and accuracy were determined for the proposed method. The proposed sample preparation and the developed chromatographic method using hydrophilic interaction liquid chromatography (HILIC) were successfully applied for the determination of glycerol, propylene glycol, and nicotine in refill liquid samples. For the first time, the developed method using HILIC-MS/MS has been applied for the determination of the main components of refill liquids in a single analysis. The proposed procedure is rapid and straightforward and is suitable for quick determination of glycerol, propylene glycol, and nicotine. The nicotine concentrations corresponded to the labelling of samples (it varied from <LOD—11.24 mg/mL), and the ratios of propylene glycol to glycerol were also determined.