Synthetic peptides, with a single or multiple modification sites, are widely used as substrates to study protein modification enzymes, such as kinases and phosphatases 1-5. However, unlike protein substrates, peptides with a small molecular mass and a low binding affinity to matrices have limited use for certain assays. For example, peptides are not suitable for western blot and often display low sensitivity in ELISA 6-9. In this protocol, we describe a method that utilizes phosphopeptides to produce ligated phosphoprotein (LPP) substrates for the study of protein phosphatases 8, 10, 11. Synthetic phosphopeptides were easily ligated through a carboxyl-terminal reactive thioester of an intein-generated carrier protein (CP) to yield the LPP substrates. These substrates were used for immobilization and ELISA analysis on a 96 well microtiter plates 12. This approach circumvents the problems associated with inefficient and variable binding by small peptides to polystyrene microtiter plates, thus resulting in significantly higher sensitivity and consistency, when compared to the use of free peptide substrates.