Anti-IDs were generated through creation of hybridomas from immunizations of mice with the Fab fragment of the therapeutic mAb. Multiple characteristics of the anti-IDs including the binding kinetics, affinities for the mAb and epitope binning were determined to attempt to triage the number of Anti-IDs to be screened. The results of the binding kinetics and affinities showed similar characteristics for all anti-IDs generated in the present study, and all of these anti-IDs were found to be in the same epitope bin. These results required all 18 anti-IDs to move forward to be screened to determine the optimal reagents for the PK assay. However, these results did not account for the effects of matrix and/or soluble antigen interference on the ability of the anti-IDs to bind to the mAb. In addition, hybridomas often lead to a large number of clones and it is inefficient and costly to label and screen all clones. Therefore, a two-step strategy was developed to identify optimal reagents and assay format to differentiate and select anti-IDs using an ECL screening assay and then a labeled anti-ID sandwich ECL screening assay with a GUI to do multiple scoring parameters analysis. This strategy was successful in identifying the most robust PK assay format with the optimal reagents for a mAb.