A 96-plex homogeneous PEA immunoassay was developed to improve high-throughput detection of protein biomarkers. The assay uses a modified design with fewer pipetting steps and improved intra-assay precision, a new enzymatic system with a hyper-thermostable enzyme (Pwo) for enhanced sensitivity, and an inter-plate control and normalization procedure for improved inter-assay precision. The assay performed well in complex samples such as serum, plasma, and xenografted mice, requiring only 1 μL of sample per test. The PEA probes were generated by coupling antibodies to unique oligonucleotides, which are extended by a DNA polymerase to form a unique sequence that acts as a surrogate marker for the specific antigen. The assay was validated using 92 cancer-related proteins and showed high sensitivity, specificity, and scalability. The PEA was also tested on dried blood spots and sera from xenografted mice, demonstrating its ability to work with low sample consumption. The assay was found to be highly specific, with no unspecific antibody binding events or assay interference. The PEA was also shown to be scalable, with a high correlation between 24-plex and 96-plex results. The assay was further validated for its ability to detect human proteins in sera from xenografted mice and in human dried blood spots. The PEA was found to be highly compatible with DBS sampling, which requires minimal blood volume and is well-suited for infant screening. The study concludes that the 96-plex PEA immunoassay is a robust and scalable platform for high-throughput protein marker discovery and research.A 96-plex homogeneous PEA immunoassay was developed to improve high-throughput detection of protein biomarkers. The assay uses a modified design with fewer pipetting steps and improved intra-assay precision, a new enzymatic system with a hyper-thermostable enzyme (Pwo) for enhanced sensitivity, and an inter-plate control and normalization procedure for improved inter-assay precision. The assay performed well in complex samples such as serum, plasma, and xenografted mice, requiring only 1 μL of sample per test. The PEA probes were generated by coupling antibodies to unique oligonucleotides, which are extended by a DNA polymerase to form a unique sequence that acts as a surrogate marker for the specific antigen. The assay was validated using 92 cancer-related proteins and showed high sensitivity, specificity, and scalability. The PEA was also tested on dried blood spots and sera from xenografted mice, demonstrating its ability to work with low sample consumption. The assay was found to be highly specific, with no unspecific antibody binding events or assay interference. The PEA was also shown to be scalable, with a high correlation between 24-plex and 96-plex results. The assay was further validated for its ability to detect human proteins in sera from xenografted mice and in human dried blood spots. The PEA was found to be highly compatible with DBS sampling, which requires minimal blood volume and is well-suited for infant screening. The study concludes that the 96-plex PEA immunoassay is a robust and scalable platform for high-throughput protein marker discovery and research.