Ligand-binding assays are used to determine concentration levels of biopharmaceuticals in biological matrices. The whole molecule does not serve as a basis for quantification, but subregions are captured and detected by specific binding critical reagents that have been produced for the sole purpose of bioanalysis. An assay can be designed to measure the free or the total analyte. Depending on the format of the assay, different observations and interpretations could be deemed. In the case studies presented in this article, the same serum samples were subjected to analysis in parallel by two different assay formats. In three out of the four cases presented, the results and the data interpretation were remarkably different. Therefore, it is essential for the bioanalyst to communicate to other stake-holders, such as toxicologists and pharmacokineticists, what the assay detects and measures for plausible data interpretation and implication.