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SignalP is the currently most widely used program for prediction of signal peptides from amino acid sequences. Proteins with signal peptides are targeted to the secretory pathway, but are not necessarily secreted. After a brief introduction to the biology of signal peptides and the history of signal peptide prediction, this chapter will describe all the options of the current version of SignalP and the details of the output from the program. The chapter includes a case study where the scores of SignalP were used in a novel way to predict the functional effects of amino acid substitutions in signal peptides.