Bitter taste, an organoleptic characteristic of virgin olive oil, has been related to phenolic compound composition. The usual method to assess this attribute is by a sensorial panel of tasters, while in the laboratory; methods based on physicochemical properties have been assayed as K225, the most widely used one. However, a direct determination of bitterness in virgin olive oil is useful for quality-control purposes. The proposed method is supported by the observable spectral change undergone by the compounds responsible for bitterness as pH varied. This measurement was carried out directly in the oil, without prior isolation of bitter analytes. The difference of absorbance between alkaline and neutral medium showed a highly significant correlation (r = 0.988, p < 0.0001) with the conventional parameter (K225). The method was rapid, required a small sample, allowed direct determination of bitterness in virgin olive oil, and could be easily automated.