Objective <p>This study assesses the comparative performance of Isotope Ratio Mass Spectrometry (IRMS) and conventional techniques in detecting honey adulteration. Standard analytical methods, in accordance with ISIRI guidelines, were evaluated alongside δ¹³C isotopic analysis via IRMS across a dataset of 20 honey samples. Conventional analytical techniques were employed to evaluate parameters such as pre-hydrolysis reducing sugars, sucrose content, the fructose-to-glucose ratio, proline, and hydroxymethylfurfural (HMF). The advanced IRMS technique was utilized to determine δ¹³C values for glucose with high accuracy and sensitivity. The carbohydrate profile of honey, including glucose, fructose, disaccharides, and trisaccharides, was analyzed using LC/IRMS. A comparative assessment of both methods was conducted on twenty honey samples, following ISIRI guidelines.</p> Results <p>While conventional methods met ISIRI criteria in 18 out of 20 samples, IRMS produced contrasting findings—only 2 samples were identified as genuine and free from adulteration. Statistical evaluation using Prism software revealed a significant discrepancy between the two techniques, with a p-value &lt; 0.05, indicating stronger confidence in IRMS results. The findings highlight the necessity of reconsidering current protocols for honey adulteration detection in order to preserve the purity and credibility of this high-value natural product.</p> Graphical Abstract <p></p>