This research aimed to examine the chromatographic behaviour of tramadol hydrochloride using the hydrophilic interaction liquid chromatography (ZIC-HILIC) method. The chromatographic analyses were performed using two lab-made HILIC columns, ZIC-2 and ZIC-3, which differ in spacer lengths between the ionic site groups. The research specifically explores the influence of these different spacer lengths on the retention behaviours of tramadol. The investigation focused on analyzing various separation parameters, including acetonitrile content, buffer concentration, and pH of the eluents. The mobile phase used in this study was a mixture of acetonitrile and acetate buffer with a pH of 5 and ionic strength of 25 mM, in a ratio of 90:10 (v/v). Elution was performed using a flow rate of 0.5 mL/min, employing an isocratic method. The compound was detected using a UV detector configured to operate at a specific wavelength of 273 nm. The results demonstrate that the ZIC-HILIC mode proves to be a simple and efficient approach for identifying tramadol in drugs. The HILIC techniques proposed in this study showed excellent precision (RSD below 1%) and a robust linear correlation between the calibration graph and tramadol concentration. The tramadol concentration range was determined to be 0.1-6.3µg/mL. The HILIC methods demonstrated excellent LOD of 0.080 and 0.050 µg/mL and LOQ ranging from 0.242 to 0.151µg/mL for tramadol in ZIC-2 and ZIC-3, respectively. The main percentage recoveries of tramadol were (98.80%-100.33%) and (98.60%-101.33%) in ZIC-2 and ZIC-3, respectively.