Olives are a potential source of natural anti-oxidants; unfortunately, a complete characterization of olive fruit phenolic profile is still to be achieved, as it is characterized by great complexity and variability. In this study, electrospray ionization mass spectrometry (ESI-MS) was used to characterize phenolic extracts from olive fruits, establishing the most suitable conditions of analysis in order to detect phenolic derivatives. This technique proved to be very useful in the identification and in the structure elucidation of olive phenolic compounds. The olive pulp phenolic fraction proved to be dominated by phenolic glucosides, mainly hexose derivatives of phenolic alcohols, flavonoids, secoiridoids and oleosides. Several oleuropein derivatives were detected, proving that they are already present in olive pulp and not, as previously suggested, originated during oil extraction. With this study an effective method to characterize olive phenolic compounds was offered and it was possible to confirm the presence of several biophenols in olive extracts, thus giving a contribution to the characterization of olive pulp phenolic fraction.