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MicroRNAs (miRNAs) are small non-coding RNAs that post-transcriptionally modulate gene expression and orchestrate a wide range of biological and pathological processes. The use of high-throughput screening technologies, in particular microscopy-based screenings (also known as high-content screenings), coupled with genome-wide libraries for modulation of miRNA levels, allow for comprehensive functional analysis of each member of the miRNome in different phenotypic cell-based assays. The wealth of information obtained from such screenings spans across various fields of research, including cancer, cardiovascular, cell reprogramming, and infection biology. Here, we provide an overview of the rationale for performing screenings using synthetic libraries of miRNA mimics and inhibitors, and of the microscopy-based miRNA screenings performed to date. Moreover, a list of resources available for such endeavor is provided. Finally, we describe a detailed procedure for a case study where microscopy-based screening using a library of miRNA mimics was performed to identify miRNAs that control infection of epithelial cells by the bacterial pathogen Salmonella. The methodologies described here can be easily adapted for screenings addressing other biological questions., (Copyright © 2018 Elsevier Inc. All rights reserved.)