Background: It has previously been shown that increased levels of plasma tissue inhibitor of metalloproteinase 1 (TIMP-1) is associated with shorter survival for patients with colorectal cancer (CRC). Furthermore, plasma TIMP-1 levels have been found to be elevated in patients with early-stage CRC. Objective:It was the aim of this study to develop a new dual monoclonal antibody (mAb) sandwich immunoassay for TIMP-1 in order to achieve better resolution of non-cancer and cancer plasma specimens. Methods: Chemiluminescence immunoassay techniques were used to screen 240 combinations of TIMP-1 mAbs for their ability to interact with each other and to allow for further characterization of the sandwiching antibody pairs. Five mAb pair combinations were selected for assessment of their ability to resolve non-cancerous and cancerous plasma specimens by TIMP-1 measurement. Based on this testing, a final assay format was chosen for further validation. The results for the final assay were compared with measurements obtained in a TIMP-1 ELISA that had previously demonstrated the ability to resolve healthy blood donors and CRC specimens. Results: The clinical results support that the new dual monoclonal immunoassay has statistical discrimination equivalent to the ELISA. Additionally, the immunoassay had a high reproducibility and specificity. Conclusion: The clinical evaluation of five TIMP-1 immunoassays resulted in the development of a new immunoassay. The new TIMP-1 immunoassay showed superior analytical performance to our previously used ELISA.