Although various reports pointed to 6-methoxymellein (1) as a key player imparting the bitter taste in carrots, activity-guided fractionation experiments recently gave evidence that not this isocoumarin but bisacetylenic oxylipins contribute mainly to the off-taste. Among these, (Z)-heptadeca-1,9-dien-4,6-diyn-3-ol (2), (Z)-3-acetoxy-heptadeca-1,9-dien-4,6-diyn-8-ol (3), and (Z)-heptadeca-1,9-dien-4,6-diyn-3,8-diol (falcarindiol, 4) have been successfully identified. In the present study, an analytical procedure was developed enabling an accurate quantitation of 1-4 in carrots and carrot products. To achieve this, (E)-heptadeca-1,9-dien-4,6-diyn-3,8-diol was synthesized as a suitable internal standard for the quantitative analysis of the bisacetylenes. On the basis of taste activity values, calculated as the ratio of the concentration and the human sensory threshold of a compound, a close relationship between the concentration of 4 and the intensity of the bitter off-taste in carrots, carrot puree, and carrot juice was demonstrated, thus showing that compound 4 might offer a new analytical measure for an objective evaluation of the quality of carrot products. Quantitative analysis on the intermediate products in industrial carrot processing revealed that removing the peel as well as green parts successfully decreased the concentrations in the final carrot puree by more than 50%.