Many widely-used non-steroidal anti-inflammatory agents (NSAIDs), e.g. ibuprofen, are extensively metabolised as their acyl glucuronides (AGs), and the reactivity of these AGs raises important questions regarding drug safety and toxicity. In order to understand better the structure-reactivity of these metabolites, we have performed a detailed study of the synthesis, structural analysis and computed transacylation reactivity of a set of acyl glucuronides (AGs) of phenylacetic acids with varying alpha-substitution. A selective acylation procedure was used to prepare all the desired 1-(phenyl)acetyl-beta-D-glucopyranuronic acids 9, 12, 13 and 15 as single 1beta-anomers in good yields. Their reactivity was measured using 1H NMR spectroscopy in pH 7.4 buffer: the dominance of transacylation over hydrolysis in this system was confirmed together with the measurement of half-lives of the 1beta-isomers of the AGs. The half-lives ranged from 20 min for compound 9 to 23 h for 15. The lack of any significant concentration dependence of the reactivity suggests that the main mechanism is intramolecular. A novel computational chemistry and modelling study was performed on both the ground states of the AGs and the transition states for acyl migration to search for correlations with the kinetic data and to probe the mechanistic detail of the acyl transfer. An excellent degree of correlation was found between the calculated activation energies and the rates of transacylation. Especially, transition state analysis provided for the first time a firm mechanistic explanation for the slower kinetics of the (S)-isomer AG 13 compared to the (R)-isomer 12, thus throwing important light on the pharmacokinetic behaviour of marketed NSAIDs.