Protein kinase have remained attractive drug targets in the treatment of various pathological conditions with recent attention moving from ATP-competitive inhibitors towards the discovery and development of peptide inhibitors targeting the protein kinase substrate-binding site. Initially discovered in yeast two-hybrid screen, PYC98 is a JNK inhibitory peptide with a 5-fold increased potency to inhibit c-Jun phosphorylation in its D- retroinverso form. In vitro JNK activity assays revealed that D-PYC98 inhibited phosphorylation of an EGFR-derived peptide substrate, transcription factors c-Jun and ATF2, and the microtubule-regulatory protein DCX (doublecortin), but not phosphorylation of the transcription factor Elk1 or the microtubule-destabilising proteins SCG10 and Op18 (Stathmin). Surface Plasmon Resonance analysis confirmed D-PYC98 binding to both non-phosphorylated (inactive) and active JNK1, but not the substrates c-Jun and Elk1. Further biochemical analyses to determine the kinetics of inhibition revealed the non-ATP competitive mechanism of action of D-PYC98. The targeting the common docking site of JNK1 by D-PYC98 was further confirmed by BIAcore competition assay in presence of TIJIP and the failure of JNK1 common docking site mutants (JNK1-R127A-E329A) to bind D-PYC98. Lastly, in evaluating the efficacy of this peptide to act as a substrate competitive inhibitor in cell culture system, we observed the action of a cell-permeable version of D-PYC98 (D-PYC98-TAT) to inhibit c-Jun phosphorylation at Ser63 under hyperosmotic stress conditions. Overall, D-PYC98 is a novel substrate-selective, non-ATP competitive inhibitor of JNK, and in its modified form as D-PYC98-TAT represents an effective JNK-inhibitor suitable for use as a probe to discern particular biological functions of this important stress-responsive kinase in cellular systems.