Abstract: Jingzhaotoxin VIII and Huwentoxin VI are two kinds of spider peptidetoxins. The most of amino acid sequences of the two peptidetoxins were determined by Edman degradation, with a few of uncertain sequences due to the ambiguous signals in Edman degradation. In order to identify the uncertain parts of the sequences, the two toxins were digested by trypsin and then analyzed by electrospray ionization Qq time of flight mass spectrometry (ESI Qq TOF MS). By interpreting the resulting MS and MS/MS spectra related to the uncertain sequences with analytical software and by hand, the amino acid sequences of uncertain parts were clearly identified. The research shows that we have got the whole sequences of HWTX I and JZTX VIII. Compared with contraditional methods, the de novo sequencing using mass spectrometry has particular advantages in peptide structure analysis and identification of proteins and peptides, and can be used as a good complement for Edman degradation.