High-throughput protein assays are crucial for modern diagnostics, drug discovery, proteomics, and other fields of biology and medicine. It allows simultaneous detection of hundreds of analytes and miniaturization of both fabrication and analytical procedures. Photonic crystal surface mode (PC SM) imaging is an effective alternative to surface plasmon resonance (SPR) imaging used in conventional gold-coated, label-free biosensors. PC SM imaging is advantageous as a quick, label-free, and reproducible technique for multiplexed analysis of biomolecular interactions. PC SM sensors are characterized by a longer signal propagation at the cost of a lower spatial resolution, which makes them more sensitive than classical SPR imaging sensors. We describe an approach for designing label-free protein biosensing assays employing PC SM imaging in the microfluidic mode. Label-free, real-time detection of PC SM imaging biosensors using two-dimensional imaging of binding events has been designed to study arrays of model proteins (antibodies, immunoglobulin G-binding proteins, serum proteins, and DNA repair proteins) at 96 points prepared by automated spotting. The data prove feasibility of simultaneous PC SM imaging of multiple protein interactions. The results pave the way to further develop PC SM imaging as an advanced label-free microfluidic assay for the multiplexed detection of protein interactions.