This study outlines the synthetic and purification process used to obtain hyperbranched polyglycerol (HPG) within a molecular weight range not previously obtainable. Purified fractions with low polydispersity are achieved via successive precipitations from acetone. Furthermore, the use of the initiator molecule 5-hexyne-1-ol introduces alkyne functionality at the core of each HPG molecule. Alkyne-azide cycloaddition chemistry in combination with NMR and field flow fractionation analysis is used to verify the availability of the alkyne core for bioconjugation across the range of molecular weights.