Kallikrein-related peptidase 6 (KLK6) is a serine protease, and one of fifteen kallikrein members located on chromosome 19. KLK6 is associated with the regulation of axonal growth following spinal injury, tumor cell metastasis, and alpha synuclein aggregate pathologies like Parkinson's, making KLK6 a potentially important biomarker. We generated a KLK6 expression vector for large-scale expression in Escherichia coli. After affinity chromatography purification and SDS-PAGE confirmation, mice were immunized with the purified protein for development of monoclonal B cell populations. Our immunizations generated five hybridomas (1D8, 2E3, 3B7, 5G8, and 5H12) against KLK6. Isotyping analysis revealed that clone 2E3 was IgG2b, while the other four clones were IgG1. Further studies found that clone 5G8 could be used to visualize specific KLK6 bands by Western blot analysis from ovarian cancer patient serum and plasma, and also in mouse liver lysates. Finally, we established a sandwich ELISA pair and determined their sensitivity for KLK6 to be 10 ng/mL. These findings establish an essential tool for the detection and analysis of KLK6.