The profile of phenolic compounds in strawberry fruits varies significantly among cultivars. High performance liquid chromatography with diode array detection coupled to electrospray ionization mass spectrometric detection with positive and negative modes of ionization was employed to identify phenolic compounds in extract of strawberries from Charlotte cultivar. This is the first time phenolic profile of Charlotte cultivar has been characterized. The fruits contained phenolics belonging to six groups: anthocyanins, ellagic acid and its conjugates, gallotannins, flavonols, flavanols and hydroxycinnamic acid derivatives. The presence of pentagalloyl glucose in strawberries fruits was reported for the first time.