The selection of suitable antibodies is a critical step in immunoassay development, since the final assay performance is predetermined by this decision to a large extent. Particularly, the screening for matching pairs in sandwich immunoassays is difficult, if both antibodies are derived from one species or when monoclonal antibodies are only available as cell supernatants. Several microplate-based approaches for in situ labeling of detection antibodies were tested, in order to avoid time consuming purification of antibodies for enzyme conjugate synthesis. We investigated labeling with anti-species antibodies and Fab fragments thereof, labeling with protein G and biotinylation of cell supernatants without prior purification. Antibodies against peanut proteins were used as a model and signal-to-blank ratios were used in all cases as a measure of the antibody pair performance. Amongst the investigated approaches, preincubation of the detection antibody with labeled anti-species antibody turned out to be most suitable under our conditions. Diagrams, showing the performance of all possible antibody combinations, were generated with this method and were compared to results obtained with covalently labeled detection antibodies. Finally, a flowchart is presented, suggesting an efficient strategy for the development of highly sensitive sandwich immunoassays.