Two novel disulfide-rich peptides were isolated and purified from the marine spongeStryphnus ponderous collected from the coast of Catalonia, Spain. Mass spectroscopic analysis showed monoisotopic masses of 3376.36 Da [M+H]+ and 3389.64 Da [M+H]+ for the peptides SP-844 and SP-848 respectively. The two peptides were found to contain three disulfide bonds and preliminary 1H NMR experiments indicated that the two peptides are likely structurally very similar, consisting of similar sequences. LC-MS/MS De Novo sequencing utilizing reduction/alkylation protocol followed by enzymatic cleavage enabled the peptide sequences to be partially determined. To investigate the potential bioactivities of Stryphnus peptides, SP-844 and SP-848 were screened for enzyme inhibitory activity (trypsin and bacterial sialidase) and found to be inactive against both enzymes. On the contrary, previously characterized disulfide-rich peptides, SF-834, SF-838, and SF-880 isolated from Stryphnus fortis collected from Northern Norway (300m depth) were found to exhibit bacterial sialidase (Clostridium perfringens) inhibitory activity. A liquid chromatography/tandem mass spectrometry (LC-MS/MS) with multiple reaction monitoring (MRM) method was developed for quantitation of SF-834 in rat plasma. Unifi information system was used to process the chromatograms and to validate the method. The method was found to be specific and sensitive with a lower limit of quantitation (LLOQ) of 100ng/ml. Linearity and repeatability of the method were confirmed for the range 100-10,000 ng/with a coefficient of correlation (R2) of >0.995. The precision for the quality control (QC) samples of 8000 and 800 ng/ml was found to be below 12%. The accuracy of the method was partially validated for QC level of 8000 ng/ml which showed accuracy above 96.7% (±9% of the nominal concentration) for four replicates and two replicates showed accuracy above 20% of the nominal concentration. The QC level of 800 ng/ml showed accuracy above 86% (-14% of the nominal concentration) for only two replicates and an accuracy range of 67.5% to 77.9 % for the other four replicates. In the Unifi information system, the coefficient of variation (CV%) for standards 100, 2000,5000, and 10,000 ng/ml were below 15%. While the CV% for the standards of 200, 500 and1000 ng/ml concentrations were above 20%. Since the CV% was found to be acceptable for the range 2000-10,000, the SF-834 stability samples (2000 and 8000 ng/ml) in rat plasma were analyzed and processed in Unify to quantify the SF-834 concentrations and the results showed that SF-834 was extremely stable in rat plasma.