Cross-linking mass spectrometry (XL-MS) is an emerging technique to obtain structural information of biomacromolecules and their complexes in cells and in vitro. In particular, certain photo-reactive amino acids (pA) such as photo-leucine (pLeu) and photo-methionine can provide unique short-distance information on structural core regions of proteins. Here, we present a protocol for high-yield incorporation of pLeu in proteins recombinantly expressed in Escherichia coli. The protein is expressed at high cell densities, which reduces the required amount of the photo-reactive amino acid by a factor of 10 compared to standard protocols, while maintaining high incorporation rates. For the two chaperones Trigger Factor and SecB, up to 3 mg of photo-leucine labeled protein were thus obtained from 100 ml of cell culture, with label incorporation rates of up to 34%. For Trigger factor, UV-induced cross-linking lead to the identification of 12 cross-links that are in agreement with published three-dimensional structures. The accessibility of milligram amounts of photo-leucine-labeled proteins at low costs will be highly useful to address structural biology questions.