Studies of protein structure and function using single-molecule fluorescence resonance energy transfer (smFRET) benefit dramatically from the ability to site-specifically label proteins with small fluorescent dyes. Genetically encoding the unnatural amino acid (UAA) p-acetylphenylalanine is an efficient way to introduce commercially available fluorescent tags with high yield and specificity. This protocol describes the expression in Escherichia coli of proteins containing this UAA in response to the amber stop codon TAG. Proteins were purified with high yield and subsequently labeled with the hydroxylamine derivative of Alexa Fluor® 488 functioning as a fluorescent donor dye. The proteins were then labeled via maleimide coupling chemistry at a unique cysteine with the acceptor dye Alexa Fluor® 594 to yield a dual-labeled protein ready for subsequent smFRET observation.