Display omitted •This work presents the most updated procedure to quantify bitter acids in hops.•It was developed considering traditional and sustainability parameters.•Complete separation of seven targeted compounds was achieved by UHPLC.•Much higher sample throughput was achieved when compared with reference methods.•Undesirable diethyl ether and methanol were replaced with dimethyl carbonate. The main precursors of bitter principles of beer are α-acids, followed by β-acids. Determination of their content in hop cones and their derivatives is an essential part of brewing. HPLC-UV is the preferred technique when a more precise quantification of some specific bitter acids are required. However, current methods do not allow the proper quantification of all the six major α- and β-acids; furthermore, these multi-step methods generate considerable volumes of chemical waste. In this work, a new UHPLC-UV protocol was developed and compared side-by-side with reference, considering traditional and sustainability parameters. Baseline separations were achieved for the six major bitter acids and xanthohumol, while a greener and faster sample preparation procedure was developed. The new validated procedure could be adopted for quality control of hops and their derivative products with enhanced sample throughput and with a potential gain in the safety of the analyst.