Urokinase plasminogen activator (uPA) is a serine protease that activates plasminogen to plasmin in the blood fibrinolytic system. It is also implicated in events related to cell invasion/migration. Plasminogen activator inhibitor type 1 (PAI-1) is involved in the regulation of the blood fibrinolytic system and forms a 1:1 covalent complex with tPA and uPA. The sensitive quantitative measurement of total human PAI-1 uPA complex antigen in plasma, serum, urine, cell culture media, or tissue extract samples is easily performed with this 96 well strip format ELISA kit. Concentrations of PAI-1 uPA complex in normal human plasma are low. The median concentration of complex in plasma samples from breast cancer patients was 0.068 ng/ml. Complex concentration in primary breast cancer tumor tissue extracts varied from 0.22-5.3 ng/mg total protein and were prognostic for decreased tumor size and grade and increased survival. Levels were associated with adverse grade and poor survival in a separate study. Values for lung cancer tumor tissue extracts were similar to breast cancer and varied from 0.11-5.74 ng/mg total protein. The assay measures PAI-1 uPA complex in the 0.1-100 ng/ml range. Samples with complex levels above 100 ng/ml should be diluted in plasma or similar fluid devoid of PAI-1 or uPA. Human uPA will bind to the capture antibody coated on the microtiter plate. After appropriate washing steps, anti-human PAI-1 primary antibody binds to the captured protein. Excess primary antibody is washed away and bound antibody is reacted with the secondary antibody conjugated to HRP. Following an additional washing step, TMB substrate is used for color development at 450 nm. Color development is proportional to the concentration of PAI-1 uPA complex in the samples. A standard calibration curve is prepared in PAI-1 depleted plasma using dilutions of purified PAI-1 uPA complex and is measured along with the test samples. All reagents and standards are provided in these ELISA kits.