Accurately measuring the brain concentration of a neurotherapeutic is critical in determining its pharmacokinetic profile . Biologics are potential therapeutics for neurologic diseases and biologics fused to an antibody targeting a transcytosis receptor at the Blood-Brain Barrier, designated as antibody-biologic fusion proteins, are Blood-Brain Barrier penetrating neurotherapeutics. The use of sandwich immunosorbent assays to measure concentrations of antibody-biologic fusion proteins in brain homogenates has become increasingly popular. The raw brain homogenate contains many proteins and other macromolecules that can cause a matrix effect, potentially interfering with the limit of detection of such assays and reduce the overall sample signal. Further, the low sample loading volumes while running these assays can reduce the sample signal. Our aim was therefore to optimize the existing tissue sample preparation and processing to concentrate the sample to elevate the signal of the analyte. Here, we present a protocol for concentrating and increasing the signal of transferrin receptor antibody-biologic fusion proteins in mouse brain homogenates using the Amicon Ultra Centrifugal filters. • The presented method uses the Amicon Ultra Centrifugal filters to concentrate mouse brain tissue homogenates. • The concentrated brain tissue homogenates are then assayed using standard sandwich enzyme-linked immunosorbent assay (ELISA) protocols. • This method improves upon the traditional brain homogenization procedure and ELISA measurements for antibody-biologic fusion proteins by effectively concentrating brain tissue homogenates.