Lysine methylation of non-histone proteins is a rapidly emerging field in proteomics and cellular signaling. Therefore, mapping lysine methylation sites and identifying the responsible protein lysine methyltransferases (PKMTs) are important scientific challenges and many groups are working on these subjects. However, there are several instances in which initially reported novel PKMT substrates could not be validated in further research. To help avoiding erroneous identification of PKMT substrates, we have collected a list of biochemical recommendation and guidelines in this review that should be applied in projects searching for novel PKM substrates. They are based on general principles governing the peptide and protein interaction of PKMTs, properties of PKMT assays and an analysis of the most critical factors in previous studies. We are confident that strict application of these rules can help to increase the quality of research in this developing field.