The proactive generation of anti-idiotypic antibodies (anti-IDs) against therapeutic antibodies with desirable properties is an important step in pre-clinical and clinical assay development supporting their bioanalytical programs. Here, we describe a robust platform to generate anti-IDs using rabbit single B cell sorting-culture and cloning technology by immunizing rabbits with therapeutic drug Fab fragment and sorting complementarity determining regions (CDRs) specific B cells using designed framework control as a negative gate to exclude non-CDRs-specific B cells. The supernatants of cultured B cells were subsequently screened for binding to drug-molecule by enzyme-linked immunosorbent assay and the positive hits of B cell lysates were selected for cloning of their immunoglobulin G (IgG) variable regions. The recombinant monoclonal anti-IDs generated with this method have high affinity and specificity with broad epitope coverage and different types. The recombinant anti-IDs were available for assay development to support pharmacokinetic (PK) and immunogenicity studies within 12 weeks from the start of rabbit immunization. Using this novel rapid and efficient in-house approach we have generated a large panel of anti-IDs against a series of 11 therapeutic antibody drugs and successfully applied them to the clinical assay development.