The study presents the development of a 96-plex proximity extension assay (PEA) for high-throughput detection of protein biomarkers. The PEA is based on a modified design that reduces pipetting steps and improves intra-assay precision, an enzymatic system using a hyper-thermostable enzyme (Pwo) for room temperature reagent addition, and an inter-plate control and normalization procedure for improved inter-assay precision. The multiplex PEA was found to perform well in complex samples such as serum, plasma, xenografted mice, and dried blood spots, consuming only 1 μL of sample per test. The technique demonstrates high sensitivity, specificity, and scalability, making it suitable for large-scale data generation and personalized health care based on molecular biomarkers.The study presents the development of a 96-plex proximity extension assay (PEA) for high-throughput detection of protein biomarkers. The PEA is based on a modified design that reduces pipetting steps and improves intra-assay precision, an enzymatic system using a hyper-thermostable enzyme (Pwo) for room temperature reagent addition, and an inter-plate control and normalization procedure for improved inter-assay precision. The multiplex PEA was found to perform well in complex samples such as serum, plasma, xenografted mice, and dried blood spots, consuming only 1 μL of sample per test. The technique demonstrates high sensitivity, specificity, and scalability, making it suitable for large-scale data generation and personalized health care based on molecular biomarkers.