The early detection and diagnosis of cancer lies central to successful treatment and improved patient outcome. Current techniques are limited by the nature of the biological receptor and the assays available. This paper reports the use of novel biological probes, peptide aptamers, in detecting cyclin-dependent protein kinases (CDKs) whose activity is important in proliferating and cancerous cells. We describe, specifically, the optimization of an orientated peptide aptamer surface and its utilization in establishing a highly specific, low-nanomolar sensitive, detection protocol for the active form of CDK2. In comparing target binding affinity of two different aptamers (pep6 and pep9), both constructed through the insertion of peptide sequences into the surface of a scaffold protein, one was observed to be consistently more effective. Significantly, the pep9 aptamers were able to detect subtle changes in the conformation of CDK2 associated with activation of its catalytic activity that may be caused by the phosphorylation of a single amino acid (threonine 160). A typical response toward the inactive form of CDK2 was in the range of 0.5-2% of the binding of the active form of CDK2 in the concentration range from 2 to 20 nM. Although antibodies are occasionally able to recognize conformations in their targets, this is the first time that a nonantibody protein probe has been used to detect an active protein isoform. Because peptide aptamers are usually raised against full-length proteins, this raises the possibility that peptide aptamers will be able to extend the repertoire of probes that recognize protein conformations, post-translational modifications (PTMs), or conformations stabilized by PTMs.