We report a detailed characterisation of the preparation of 5,6-cellulosene, a poorly investigated reactive cellulose derivative. A key literature procedure is followed, which utilises partially hydrolysed technical cellulose acetate, as a 6-OH rich starting material. Thorough NMR analysis, using well-established solution-state NMR protocols, is performed to identify mechanisms of reactivity. The major drawback and potential opportunity identified in this study is the propensity for 5,6-cellulosene linkages (containing an alkenyl-acetal moiety) to undergo hydrolysis, leading to depolymerisation. However, now we demonstrate, using modern theoretical and NMR methods, that hydrolysis of cellulosene is still controllable under room temperature photoinitiated thiol–ene reaction conditions, and aqueous workup. The suitability of 5,6-cellulosene as a synthon towards thiol–ene chemistry is finally assessed (under thermal and photoinitiated radical addition) using a model amine-appended thiol. Clear applications of interest are in biomedical science, due to the abundance of thiol-containing proteins utilised in selective protein labelling.