<p>(<i>E</i>)-5-Methylhept-2-en-4-one is a key flavour compound in hazelnuts with practical applications in perfumery, flavour, and food industries due to its unique sensory properties. This study developed an effective and sustainable achiral reverse-phase high-performance liquid chromatography (HPLC) method with diode array detection (DAD) to determine (<i>E</i>)-5-methylhept-2-en-4-one and its reaction intermediates prepared via chiral-pool-based chemoenzymatic synthesis. Given that two reaction intermediates are thermally unstable at the high temperatures used in gas chromatography, HPLC separation at ambient temperatures (avg. 25&#xa0;°C) was an appropriate approach. Optimised HPLC conditions consisting of C18 type stationary phase, mixture of acetonitrile: water (60:40, v/v) as mobile phase under isocratic conditions and flow rate 0.5 mL.min⁻¹ were used. Chiral normal phase HPLC–DAD method with polysaccharide derivative stationary phase and <i>n</i>-hexane:propan-2-ol (90:10, v/v) as mobile phase was used for separation of enantiomers of (<i>E</i>)-5-methylhept-2-en-4-one. Both achiral and chiral methods were successfully used for monitoring the course of chemoenzymatic synthesis, conversion of reaction intermediates and purity of final product. The achiral HPLC–DAD method showed good linearity (<i>R</i>² = 0.9997) in concentration range 0.5–100 μg.mL⁻¹. The limits of detection and quantitation were 0.1 and 0.3 μg.mL⁻¹, respectively. Using developed chiral HPLC–DAD method, sufficient enantioseparation was achieved with resolution of 4.73. Values of LOD and LOQ were 0.2 μg.mL^–1 and 0.5 μg.mL^–1, respectively. Besides synthesis monitoring and purity product determination, the developed chiral and achiral HPLC methods could find application in the analysis of (<i>S</i>)-(<i>E</i>)-5-methylhept-2-en-4-one in nut-flavoured foods (e.g., pastries, cookies, desserts, beverages) and personal care products, as well as differentiate pure olive oil from those adulterated with hazelnut oil.</p> Graphic Abstract <p></p>