Caspases play a key role in apoptosis and represent important therapeutic targets for treating cancer and inflammatory diseases. In this proof-of-concept study, an electrochemical impedance spectroscopy (EIS)-based biosensor was developed for the analysis of caspase-3 activity in non-diluted biological samples using screen-printed gold electrodes (SPGE) compatible with small sample volumes (i.e. 2 mu L). A caspase-3-specific peptide substrate was immobilized on the surface of the SPGE using N-hydroxysuccinimide (NHS)-activated lipoic acid esters. The proteolytic activity of caspase-3 was analyzed using EIS, in which the presence of the enzyme resulted in cleavage of substrate peptides. Changes in the surface-immobilized substrate peptide film were detected using the apparent charge transfer resistance (R-APP) in connection with [Fe(CN)(6)](3-/4-) redox probe. The analytical performance of the biosensor was challenged with undiluted apoptotic human SH-SY5Y neuroblastoma cell lysates at 2-mu L aliquots. Control experiments were performed with healthy SH-SY5Y cell lysates and did not display a significant decrease in RAPP values. EIS data were also confirmed using a commercially available optical kit. EIS-based biosensor demonstrated a promising potential to become a versatile tool for rapid screening of caspase-3 proteolytic activity in complex biological samples. (C) 2014 Elsevier Ltd. All rights reserved.