High-performance liquid chromatography (HPLC) has been widely used as a reliable technique to quantify anthocyanins in food samples. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is a new technique that is having a great impact on food analysis. This study is the first to compare HPLC and MALDI-TOF MS quantifications of anthocyanins. The analyses were carried out for highbush blueberries at different stages of anthocyanin formation. In general, both techniques provided comparable quantitative anthocyanin profiles for the samples. HPLC could distinguish anthocyanin isomers, whereas MALDI-TOF MS proved to be more rapid in the accurate identification and quantification of anthocyanins with different masses. A single MALDI-TOF MS run took just 4 min. MALDI-TOF MS analysis can serve as a rapid alternative to HPLC for the analysis of anthocyanins in fruits.