pH-sensitive linkers designed to undergo selective hydrolysis at acidic pH compared to physiological pH can be used for selective release of therapeutics at their site of action. In this paper, the hydrolytic cleavage of a wide variety of molecular structures that have been reported for their use in pH-sensitive delivery systems was examined . A wide variety of hydrolytic stability profiles was found among the panel of tested chemical functionalities. Even within a structural family, a slight modification of the substitution pattern has an unsuspected outcome on the hydrolysis stability. This work lead us to establish a first classification of these groups based on their reactivities at pH 5.5 and their relative hydrolysis at pH 5.5 vs. pH 7.4. From this classification, four representative chemical functions belonging eand compare lysosomal cleavage in living cells..and revealed that only the most reactive functions underwent significant lysosomal cleavage, according to flow cytometry measurements. These last results question the acid-based mechanism of action of known drug release systems and advocate for the importance of in-depth structure- reactivity study, using tailored methodology, for the rational design and development of bio-responsive linkers.