Electrophoretic mobility shift assays (EMSAs) play an important role in analytical chemistry, quantitative bioscience, and point-of-care diagnostics. Emerging microfluidic lab-on-a-chip technologies bring high throughput and multiplexed analysis to affinity-based electrophoretic separations, greatly advancing the performance of traditional EMSAs. This review elaborates on the relevant theoretical basis for EMSAs, surveys microfluidic-based EMSA applications in molecular conformation analyses, immunoassays, affinity assays and genomics, and outlines challenges and potential future improvements needed from this powerful assay.