The aim of this study was to develop and validate a method for the determination of balenine/ophidine (hereafter referred to as "balenine") in whale extracts and muscle samples from Balaenoptera acutorostrata. Further, the goal was to evaluate the method's applicability for the determination of other histidine-containing dipeptides (HCDs): anserine and carnosine and their amino acids π-methylhistidine, τ-methylhistidine, histidine, and β-alanine. For balenine, the LOD and LOQ were found to be 0.03 and 0.1 mg/g, respectively, and the linear range was validated up to 160 mg/g. Trueness was evaluated by spiking experiments with balenine, and the recovery was found to be 88-90%. A comparison of the results showed that most of the other analytes were within 80-120% of the value found with the previously developed and validated method. Precision and internal reproducibility for balenine was around 0.9 and 2%, respectively, with measurement uncertainties of 2-4%. Therefore, the method was found to be fit for purpose for the determination of balenine and other HCDs and their constituent amino acids in whale meat and extracts.