In vitro enzyme assays provide sensitive and quantitative approaches to studying activity, identifying substrates or performing inhibitor analysis. These approaches can help to study enzymes that regulate different cellular processes in an organism. In addition, enzyme assays can be used to screen protein inhibitors to identify novel drug candidates. In eukaryotes, phosphatidylinositol kinase and phosphatase are enzymes that regulate the level of phosphorylated phosphatidylinositol, which controls several cellular processes. They also regulate several processes in many pathogens, such as kinetoplastid and apicomplexan parasites, and are validated drug targets. Here, we describe protocols to determine the activity of purified phosphoinositide kinases and phosphatases and screens to identify potential inhibitors. The assays are luminescence- and absorbance-based to measure the activity of phosphatidylinositol kinase and phosphatase enzymes, respectively. These assays can be adapted to other protein kinases and phosphatases.