Single-cell proteomics has the potential to decipher tumor heterogeneity, and methods such as Single-Cell ProtEomics by Mass Spectrometry (SCoPE-MS) allow profiling of several tens of single cells for more than 1,000 proteins per cell. Our laboratory has advanced state-of-the-art single-cell proteomics technology to enable linking proteomes of individual cells with phenotypes of interest. Here, we describe a microscopy-based functional single-cell proteomic profiling technology, called FUNpro, designed for general use in cultured cell lines. FUNpro enables high-throughput, real-time live-cell screening, identification, and isolation of single cells of interest, even when the phenotypes are dynamic or the cells are rare. This chapter outlines a generalized protocol for functional single-cell proteomic profiling and analysis of a standard cancer cell line, including an overview of the microscopy, cell culture, image analysis, photolabeling of cells of interest, fluorescence-activated cell sorting (FACS), and the single-cell proteomics procedures and analyses employed.