Protein kinases are an important class of enzymes and drug targets. New opportunities to discover medicines for neglected diseases can be leveraged by the extensive kinase tools and knowledge created in targeting human kinases. A valuable tool for kinase drug discovery is an enzyme assay that measures catalytic function. The functional assay can be used to identify inhibitors, estimate affinity, characterize molecular mechanisms of action (MMOAs) and evaluate selectivity. However, establishing an enzyme assay for a new kinases requires identification of a suitable substrate. Identification of a new kinase's endogenous physiologic substrate and function can be extremely costly and time consuming. Fortunately, most kinases are promiscuous and will catalyze the phosphotransfer from ATP to alternative substrates with differing degrees of catalytic efficiency. In this manuscript we review strategies and successes in the identification of alternative substrates for kinases from organisms responsible for many of the neglected tropical diseases (NTDs) towards the goal of informing strategies to identify substrates for new kinases. Approaches for establishing a functional kinase assay include measuring auto-activation and use of generic substrates and peptides. The most commonly used generic substrates are casein, myelin basic protein, and histone. Sequence homology modeling can provide insights into the potential substrates and the requirement for activation. Empirical approaches that can identify substrates include screening of lysates (which may also help identify native substrates) and use of peptide arrays. All of these approaches have been used with a varying degree of success to identify alternative substrates.