In this paper, we describe a new method for the selective extraction and quantification of glutathione (GSH) using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and maleimide-presenting gold nanoparticles (Mal-AuNPs). Our strategy utilizes the Michael addition to selectively extract GSH, from chosen samples, onto the maleimide of Mal-AuNPs. After the extraction step, the GSH bound to the AuNPs was analyzed by MALDI-TOF MS in the presence of an internal standard which was prepared by reacting Mal-AuNPs with isotope-labeled GSH ($GSH^*$). The $GSH^*$ has the same structure as GSH but a higher molecular weight, and therefore, enables absolute quantification of GSH by comparing the mass signal intensities of the GSH- and $GSH^*$-conjugated alkanethiols. Our strategy was verified by analyzing GSH-spiked fetal bovine serum and NIH 3T3 cells.