ALPK1 is an atypical protein kinase that is activated during bacterial infection by ADP-heptose and phosphorylates TIFA to activate a cell signaling pathway. In contrast, specific mutations in ALPK1 allow it to also be activated by endogenous human nucleotide sugars such as UDP-mannose, leading to the phosphorylation of TIFA in the absence of infection. This protocol describes a quantitative, cell-free phosphorylation assay that can directly measure the catalytic activity of wildtype and disease-causing ALPK1 in the presence of different nucleotide sugars. In this method, overexpressed ALPK1 is first immunoprecipitated from the extracts of ALPK1 knockout HEK-Blue cells transfected with plasmids encoding either FLAG-tagged wildtype or mutant ALPK1, and then subjected to a radioactive phosphorylation assay in which the phosphorylation of purified GST-tagged TIFA by ALPK1 is quantified by measuring the incorporation of radioactivity derived from radiolabeled ATP. Key features • Quantitative measurement of protein kinase activity of wildtype and mutant ALPK1 in the presence or absence of different nucleotide sugars such as ADP-heptose and UDP-mannose. • Cell-free experimental setup overcoming the challenge of distinguishing constitutive activity and activation by endogenous mammalian metabolites in cell-based assays. • Requires approximately 50 µg of cell extract protein/reaction, allowing up to 150 assays to be performed from an extract prepared from a single 15 cm dish of transfected cells.