Lipoproteins contain fatty acids at their amino termini through which they are anchored in lipid membranes. Here, we describe the use of alkyne fatty acids and click chemistry to label lipoproteins in bacterial cells. Exogenous fatty acids containing an alkyne group are incorporated in phospholipids and lipoproteins during bacterial growth. Protein precipitation followed by click chemistry renders lipoproteins fluorescent that can be visualized by in-gel fluorography. Alkyne phospholipids that are the acyl donor in the maturation process of lipoproteins can also be isolated from bacterial membranes for further enzymatic and kinetic studies of acyltransferase that use phospholipids as substrate.