The enzyme glucose-1-phosphate adenylyltransferase (GlgC, EC:2.7.7.27) catalyses the first step in glycogen synthesis by converting glucose-1-phosphate into ADP-glucose, which is added in turn to a growing glycogen chain by glycogen synthases. Thus far, in vitro studies of GlgC were mainly performed using colorimetric or radiolabel-based phosphate release assays, limiting the option for analysing this reaction. With this work, we present a novel in vitro continuous assay coupling the subsequent glycogen synthase reaction to the GlgC reaction, thus simulating the process of glycogen synthesis in vivo. Using this assay, we revisited GlgC catalytic parameters and screened for metabolites that affect GlgC activity in Synechocystis sp. PCC 6803. We also describe in further detail the antagonistic interplay between the GlgC activator, 3-PGA and the inhibitor, inorganic phosphate, revealing the intricate mechanism by which glycogen formation responds to fluctuations in carbon and energy supply in cyanobacteria.