An adequate bioanalytical support for a typical biotherapeutic requires a number of assays, including those to measure drug concentration and to assess induction of specific immune responses. Ligand-binding assays are the most commonly used platform in bioanalysis of biotherapeutics. Ligand-binding assays are frequently designed to detect appropriate analytes in complex biological matrices with limited or no sample pretreatment steps. The complex composition of the test matrix is highly diverse and varies from normal to disease populations. Additional post-treatment changes are often observed, including induction of antidrug antibodies. Due to potential interaction of biological matrix components, for example, rheumatoid factors, heterophilic antibodies and human anti-animal antibodies, with the test analyte or assay reagents, ligand-binding assays are often subjected to various degrees of matrix interferences that lead to an erroneous under- or over-reporting of the analyte concentration. Impact of various matrix components and practical means designed to mitigate interferences are discussed in this Review.