Dissecting the subcellular distribution of a fatty-acylated protein is key to the understanding of the molecular mechanisms regulating protein movement and function in a cell. This protocol describes how to perform single-cell imaging of palmitoylation in a fatty-acylated protein of interest with high sensitivity using click chemistry, proximity ligation and fluorescence microscopy. The initial steps in this protocol involve optimization of conditions for (i) metabolic incorporation of an alkynyl analog of palmitic acid into cellular proteins coupled with click chemistry and (ii) detecting a specific protein of interest with primary antibodies using automated fluorescence microscopy, followed by (iii) imaging palmitoylation of the target fatty-acylated protein of interest, such as Wnt, Sonic Hedgehog or H-Ras. Furthermore, we outline strategies for imaging specific fatty-acylated proteins with subcellular organelles and/or total proteome palmitoylation, and we discuss special considerations that need to be given depending on the experimental design. The use of clickable fatty acids with proximity ligation may have promising applications to the investigation of fatty acylation cell biology. The entire protocol takes ∼3 weeks to complete.