This protocol describes the in vitro phosphorylation of ubiquitin and Parkin by the kinase PINK1 using recombinant proteins. Both substrates, ubiquitin and Parkin, are phosphorylated at the conserved serine 65 residue (pS65-ubiquitin and pS65-Parkin). The protocol also includes the use of monomeric and K48- and K63-linked poly-ubiquitin chains as alternative substrates. Although there are commercially available antibodies, we have not tested their performance in this assay since, but used validated antibodies from our laboratory. An alternative antibody-independent method, the use of phostag gels to detect pS65-ubiquitin and pS65-Parkin, is described in addition.