Despite the current availability of selective inhibitors for the classical nuclear export pathway, no inhibitor for the classical nuclear import pathway has been developed. Here we describe the development of specific inhibitors for the importin alpha/beta pathway using a novel method of peptide inhibitor design. An activity-based profile was created via systematic mutational analysis of a peptide template of a nuclear localization signal. An additivity-based design using the activity-based profile generated two peptides with affinities for importin alpha that were approximately 5 million times higher than that of the starting template sequence. The high affinity of these peptides resulted in specific inhibition of the importin alpha/beta pathway. These peptide inhibitors provide a useful tool for studying nuclear import events. Moreover, our inhibitor design method should enable the development of potent inhibitors from a peptide seed.