This work proposes a practical experimental approach that allows the rapid in situ generation of a wide range of intracellular GSH concentrations in the intact hepatocyte under highly reproducible conditions. The strategy involves the use of diethyl maleate, a thiol-reactive electrophile that causes rapid and extensive GSH depletion, as well as GSH monoethylester, a GSH analogue that is readily taken up by cells and deesterified intracellularly to render GSH. For both agents, we have analyzed (i) the minimal exposure time required to produce a maximal and dose-related effect on intracellular GSH without altering hepatocyte viability or subsequent survival in culture, and (ii) the relative stability of the GSH levels achieved after removal of both modulators from the culture medium. Results show that with the appropriate timing of exposure, hepatocytes from the same preparation can be quickly synchronized to provide an extreme intracellular GSH concentration gradient (from 0.1- to 4-fold the normal liver content), which simulates an in vitro GSH dose dependency curve and remains stable for at least the subsequent 4 h of culture. The experimental design, which can be easily adapted to different cell types, provides an improved testing protocol to evaluate under reliable conditions the dose–response relationships of the thiol-redox state in a variety of biological processes. It has been applied here to investigate the role of endogenous GSH content in the covalent binding of N-acetyl-p-benzoquinoneimine, the reactive species formed during CYP-dependent bioactivation of acetaminophen, to hepatic proteins.