Prenylated proteins contain C15 or C20 isoprenoids linked to cysteine residues positioned near their C-termini. Here we describe the preparation of isoprenoid diphosphate analogues incorporating diazirine groups that can be used to probe interactions between prenylated proteins and other proteins that interact with them. Studies using synthetic peptides and whole proteins demonstrate that these diazirine analogues are efficient substrates for prenyltransferases. Photo-cross-linking experiments using peptides incorporating the diazirine-functionalized isoprenoids selectively cross-link to several different proteins. These new isoprenoid analogues should be broadly useful in the studies of protein prenylation.