A modern immunofluorescence analysis based on spectrally encoded microspheres has found numerous and constantly growing applications in disease diagnosis, environmental supervision, and fundamental science. Here we present an overview of microsphere-based methods of multiplex immunofluorescence analysis and consider such important parameters of beads, that are crucial in most microsphere-based immunoassays, as size distribution, fluorescence stability, magnetic properties, and particle material. The preparation methods of the microspheres with tunable diameter, the introduction of various types of fluorochromes, and magnetic particles into the microspheres are discussed in details. This review also addresses the advantages and disadvantages of different approaches to implement technically bead-based immunofluorescence analysis. K E Y W O R D S bioengineering, biomedical applications, functionalization of polymers, synthesis and processing techniques 1 | INTRODUCTION Bead-based immunofluorescence analysis is a powerful tool widely used in numerous applications for life science research (extracellular vesicles [EV], 1 cytokines, 2 chemokines, 3 growth factors, cells and drug research, 4 etc.) and for in vitro diagnostics 5 of infectious diseases, diabetes, cardiovascular diseases, inflammation, cancer, 6

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