Derivatives of glutathione (γ-Glu-Cys-Gly) and glutathione-derived inhibitors of enzymes involved in glutathione-related metabolism can be used as therapeutic agents against various diseases. However, their intracellular delivery is difficult because of poor cell membrane permeability due to high hydrophilicity. Conventional design strategies to enhance cell membrane permeability include the esterification of carboxy groups in γ-Glu and Gly residues. However, our design strategy involves replacing a carboxy group in the Gly residue with a structurally similar but more lipophilic counterpart; the hydrolysis rate of a Gly ester residue in glutathione is slow. Herein, to preliminarily validate our new design strategy, we investigated the cellular uptake and intracellular metabolism of carboxy group-modified glyoxalase I inhibitors derived from glutathione, which were radiolabeled for tracing their behaviors. We synthesized and evaluated three radioiodinated model compounds [¹²⁵I]2–4 using NH₂-γ-Glu[-Dab(N-(p-bromobenzoyl)-N-hydroxyl)-Gly-OH]-OH (1) as a lead compound in vitro. [¹²⁵I]2, a conventional diester, features two carboxy groups esterified with cyclopentyl alcohol. A new design strategy was followed for [¹²⁵I]3 and [¹²⁵I]4, wherein the ester group at the Gly residue in [¹²⁵I]2 was replaced by a CH₂OH or CF₃ group. These compounds showed log D_7.4 values suitable for membrane permeability. Their cellular uptake increased in a time-dependent manner; however, that of [¹²⁵I]3 remained low. While the majority of metabolites of [¹²⁵I]2 comprised multiple unknown compounds, the main metabolite of [¹²⁵I]4 was monocarboxylic acid generated by the hydrolysis of an ester of γ-Glu residue. These results provide useful insights for developing glutathione-derived therapeutic agents.