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Ranibizumab (Lucentis®), a humanized antigen-binding fragment (Fab) monoclonal antibody that blocks VEGF-A activity, is currently approved for the treatment of several retinal degenerative diseases. The assessment of drug pharmacokinetics (PK) is essential for evaluating exposure as it relates to drug safety and efficacy. For drugs administered intravitreally, systemic drug levels during the course of clinical studies are typically 100 to 1000-fold lower than those of similar therapeutics dosed intravenously, posing a significant bioanalytical challenge for PK measurements. Thus, the development of a highly-sensitive assay for measuring pg/mL levels of ranibizumab in patients' sera after intravitreal administration was needed to support clinical studies. In this report, we describe the development of a novel method that utilizes a high-affinity murine monoclonal anti-ranibizumab-VEGF-complexes antibody (MARA) reagent to measure ranibizumab in human serum. The assay format utilizes a semi-homogeneous solution phase step using a monoclonal antibody (the MARA) that binds specifically to the ranibizumab-VEGF complex, but not to either alone. This unique reagent exhibited low non-specific binding and high selectivity, increasing signal-to-noise readouts and maximizing assay sensitivity. The resulting MARA enzyme-linked immunosorbent assay (ELISA) has a lower limit of quantification of 15 pg/mL in human serum. In the assay, serum samples are incubated overnight with a mixture containing biotinylated-VEGF and MARA, which form a three-molecule complex with ranibizumab in the sample. These complexes are then captured onto streptavidin-coated wells, followed by enzymatic detection using a horseradish peroxidase-labeled-anti-murine antibody reagent and a colorimetric reaction. The assay conditions were optimized to allow for quantitative detection of "total" ranibizumab levels in serum. The assay was fully validated, establishing its high tolerance to sample matrix, as well as its suitable specificity, accuracy, dilution linearity, as well as intra- and inter-assay precision. The MARA ELISA's novel and unique approach has resulted in a considerably more sensitive ranibizumab PK assay compared to earlier versions of this assay. The MARA ELISA has been used for PK measurements in multiple ranibizumab studies, supporting this drug's life-cycle management and related preclinical and clinical-development studies., (Copyright © 2018. Published by Elsevier B.V.)