Aim: Meso Scale Discovery U-PLEX® provides an opportunity to develop multiplexed pharmacokinetic (PK) immunoassays. Two case studies demonstrate the utility of multiplexed PK methods. Materials & methods: Development of PK ligand-binding assays quantify of nonclinical plasma concentrations of a biotherapeutic that has degraded due to in vivo biotransformation, and clinical serum concentrations from two biotherapeutics spiked into a single sample. Results: Data from multiplexed U-PLEX PK methods are comparable to results from single-readout streptavidin Meso Scale Discovery gold PK methods. Multiplex measurement of a nonclinical study showed acceptable performance for accuracy, precision and dilutional linearity while a clinical study additionally passed selectivity, specificity and stability. Conclusion: Regulated, validation-ready multiplex PK methods for both nonclinical and clinical studies allow opportunities for high-throughput bioanalysis.