Single-cell analysis offers unprecedented resolution for the investigation of cellular heterogeneity and the capture of rare cells from large populations. Here, described is a simple method named interfacial nanoinjection (INJ), which can miniaturize various single-cell assays to be performed in nanoliter water-in-oil droplets on standard microwell plates. The INJ droplet handler can adjust droplet volumes for multistep reactions on demand with high precision and excellent monodispersity, and consequently enables a wide range of single-cell assays. Importantly, INJ can be coupled with fluorescence-activated cell sorting (FACS), which is currently the most effective and accurate single-cell sorting and isolation method. FACS-INJ pipelines for high-throughput plate well-based single-cell analyses, including single-cell proliferation, drug-resistance testing, polymerase chain reaction (PCR), reverse-transcription PCR, and whole-genome sequencing are introduced. This FACS-INJ pipeline is compatible with a wide range of samples and can be extended to various single-cell analysis applications in microbiology, cell biology, and biomedical diagnostics.