The aim of the present study is to develop a targeted HILIC-MS/MS method utilizing a zwitterionic HILIC column for the quantification of free CoA and short-to long-chain acyl-CoA compounds in a single analytical run (covering the full analyte range from high to low polarity) and demonstrate the utility of this method in HepG2 cell application. To achieve this, the initial method development was done on a high-resolution QTOF instrument (HRMS) for pre-screening of species and identifying their retention time. The chromatography was optimized by studying the effects of different factors such as buffer concentration and injection solvents, to obtain the satisfactory peak shape. After optimization of various LC-MS settings, a targeted method was created and validation parameters such as linearity, sensitivity, precision, recovery and matrix effect were examined to evaluate method performance in the HepG2 cells.