MALDI-peptide mass fingerprinting (PMF) and MALDI-MS/MS ion search (MASCOT- MS/MS-ion search) have become the preferred methods for high throughput identification of proteins. Unfortunately, PMF can be ambiguous, mainly when the genome of the organism under investigation is unknown and the quality of spectra generated is poor and does not allow a confident identification. The PSD fragmentation of singly charged tryptic peptide ions generated by MALDI-TOF/TOF typically results in low fragmentation efficiency and/or complex spectra, including backbone fragmentation ions (series b and y), internal fragmentation etc. Interpreting these data either manually and/or using de novo sequencing softwares can frequently be a challenge. To overcome this limitation when studying the proteome of adult Angiostrongylus costaricensis, a nematode with unknown genome, we have used chemical N-terminal derivatization of the tryptic peptides with 4-sulfophenyl isothiocyanyl (SPITC) prior to MALDI-TOF/TOF MS. This methodology has recently been reported to enhance the quality of MALDI-TOF/TOF-PSD data, allowing the obtainment of complete sequence of most of the peptides and thus facilitating de novo peptide sequencing. Proteins were extracted from adult nematodes using a solution composed of 2 M thiourea, 7 M urea and 4 % CHAPS and the analysis by 2D-electrophoresis was performed using a 18 cm linear precast IPG strip ranging from 3-10 followed by SDS-PAGE (15 % T). Several Coomassie stained spots were excised, subjected to in-gel trypsin digestion and identified by MALDI-TOF/TOF MS. When comparing samples submitted to SPITC derivatization to untreated ones, major quality enhancement was observed in corresponding peptides MS/MS spectra, resulting in an increase of both the number of peptides sequenced per sample and the length (longer and ungapped Y series) of each peptide. Concluding, when using the derivatization procedure, we have had a two-fold increase in positive identifications when using the program BLAST for searching short sequences in the nrNCBI database.