The kallikrein-related peptidases (KLKs) are implicated in prostate cancer progression via activation of growth factors, proteases and degradation of the extracellular matrix. We are investigating their potential as cancer specific biomarkers and therapeutic targets for prostate cancer. We have previously shown novel associations of KLK peptidases with processes indicative of metastasis such as epithelial-mesenchymal transition (EMT) in prostate cancer cells and chemoresistance in ovarian cancer cells. We are now establishing a comprehensive systems biology approach, in combination with 3-dimensional (3D) in vitro and in vivo models that better reflect the in vivo tumour microenvironment, to determine the KLK substrate profile or degradome, and the underlying molecular pathways, involved in these events. In particular, we are using models of prostatic bone metastasis coupled with novel proteomic approaches to define the KLK degradome in these micro-environments. Our current findings on the secretome of the bone metastatic PC3 prostate cancer cell using PICS, TAILS and PROTOMAP proteomic approaches has identified several novel substrates for KLK4 as well as those previously reported such as pro-uPA and fibronectin. In the bone matrix model, prostate cancer LNCaP cells cluster aligning with the fibrillar structure as they invade into the bone matrix in a manner typical of that seen in vivo. KLK4 proteolysis of the osteoblast-derived bone matrix has identified additional novel substrates. Analysis of the transcriptome, coupled with that of the secretome, of these model systems will provide further insight into the precise substrates that have triggered these events and the downstream signalling pathways involved. These bioengineered 3D microenvironments provide innovative models to delineate the roles of individual KLK proteases in prostate cancer development and progression.