Fluorescence-based detection of single biomolecules in solution and at room temperature has opened new avenues for understanding biological mechanisms. Single-molecule fluorescence spectroscopy (SMFS) of biomolecules requires careful selection of fluorophores, sites of incorporation, and labeling chemistries. SMFS-compatible fluorophores should permit extended, uninterrupted observations of fluorescence with high signal-to-noise ratios; more stringent considerations apply for specific methodologies, such as fluorescence resonance energy transfer and fluorescence anisotropy. Strategies for site-specific in vitro labeling of small proteins exploit the reactivity of the amino acid cysteine (Cys), allowing incorporation of one or more fluorophores; labeling of closely spaced Cys residues using bis-functionalized fluorophores allows probing of the orientation of individual protein domains. For in vitro labeling of large proteins, the options include peptide ligation, intein-mediated labeling, puromycin-based labeling, unnatural amino acid mutagenesis, and reconstitution from individual subunits or subunit fragments. For in vivo analysis, one can use proteins that are labeled in vitro and then incorporated in cells; genetic encoding of specific protein sequences can also lead to in vivo labeling, either by in vivo targeting by fluorophores or by biosynthesis of protein fusions with natural fluorophores such as the green fluorescent protein. The existing methods, along with others under development, will bring SMFS to the mainstream and advance significantly our understanding of vital biological processes.