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We have developed a technique for producing patterned biomolecule gradients on glass substrates using microstamping. The technique uses a microfabricated polydimethylsiloxane (PDMS) stamp with micrometer length features to transfer biomolecules to silane-derivatized substrates. The soaking solution for the stamp is subjected to an electric field to electrophoretically produce a gradient in solution and hence on the stamp. To test the efficacy of the procedure we tested substrates stamped with poly-D-lysine by fluorescently labeling the amine groups and measuring the fluorescence. It is possible to modify the steepness of the gradient using both voltage and electrode spacing, and the profile can be modeled using an isoelectric focusing model. Since gradients have been implicated in roles in guidance and patterning of neurons and other cell types, a flexible method to produce biomolecule gradients using microstamping may further extend the usefulness of microstamping to studies of cell patterning and guidance.