In-silico identified novel secretory signal peptides (SPs) are required in vivo to achieve efficient transfer or to prevent other cellular proteins from interfering with the process in extracellular recombinant protein (r-protein) production. 56 endogenous and exogenous secretory SPs, have been used or having the potential to be used in Pichia pastoris for r-protein secretion, were analyzed in-silico using the softwares namely SignalP4.1, Phobius, WolfPsort0.2, ProP1.0, and NetNGlyc1.0. Among the predicted 41 endogenous secretory SPs, five of them have been used in P. pastoris, and regarded as positive controls; whereas, 36 of them have not been used. Amongst, the predicted cleavage site for each of the 32 endogenous secretory SPs was found to be same by the three programs. The secretory SPs having the highest D-scores, the score quantifying the signal peptide-ness of a given sequence segment, were: MKILSALLLLFTLAFA (D=0.932), MRPVLSLLLLLASSVLA (D=0.932), MFKSLCMLIGSCLLSSVLA (D=0.918). As D-scores of these SPs are higher than that of Saccharomyces cerevisiae a-mating factor signal peptide which has been widely used for r-protein production, they can be considered as the promising candidates. Among the predicted 15 exogenous SPs, 11 have been used in P. pastoris: therefore, they were evaluated as positive controls. The three programs predicted a unique cleavage site for each of the 10 exogenous SPs; and D-scores of these SPs were within D=0.805-0.900; whereas, four exogenous secretory SPs, which have not been used in P. pastoris, have D-scores within D=0.494-0.702. (C) 2014 Elsevier Ltd. All rights reserved.