Immunoassay measurements of human kallikrein-related peptidases (KLKs) such as prostate-specific antigen (KLK3) are of great value as diagnostic indices of cancer. Despite extensive knowledge of the abundance of immunoreactive KLKs in normal and cancer-related settings, there is little information available about the proportion of immunoreactive KLK that represents active enzyme in such samples. Using KLK6 as a prototype enzyme, we have developed an assay using a serine proteinase-targeted activity-based probe coupled to antibody capture. By employing activity-based labeling, we were able to quantify the proportion of enzymatically active relative to total immunoreactive KLK6 in crude cerebrospinal fluid from routine analyses and ascites fluid from ovarian cancer patients, as well as in supernatants from cancer cell lines. Our approach allowed monitoring of pro-KLK6 conversion to its active enzyme species and demonstrated that up to 5% of immunoreactive KLK6 detected in clinical samples represents active enzyme. We suggest that this new activity-based probe assay will prove of value as a complement to routine KLK immunoassay measurements for validating KLKs as cancer biomarkers.