Gastrointestinal decontamination by activated charcoal (AC) is the most important treatment for acetaminophen (APAP) overdose. Because AC adsorbs a wide variety of toxins, it may also adsorb the oral antidote, N-acetylcysteine (NAC). NAC is a specific antidote for APAP overdose and administered as a 72-h oral regimen. We evaluated AC adsorption of NAC after APAP adsorption in vitro. Different concentrations of NAC solution diluted with simulated gastric fluids (SGF) and simulated intestinal fluids (SIF) were added to AC and incubated at 37°C for 1 h. The AC was then removed by filtration, and the NAC concentration was determined. This revealed that NAC was not only adsorbed onto the AC but also converted to N,N’-diacetyl-L-cystine (DAC), which is oxidized NAC. We then calculated the maximum adsorption capacity per gram of AC (Qm). The apparent Qm based on the amount of decreased NAC in the SGF was 400 mg/g, and that in the SIF was 714 mg/g. The actual Qm based on only the amount of adsorption in the SGF was 294 mg/g, and that in the SIF was 59 mg/g. We also determined whether or not AC could adsorb the loading and maintenance doses of NAC after APAP adsorption. The residual rate in the SGF was 2.1%, and that in the SIF was 0.3%. The rate of conversion to DAC was higher in the SIF than that in the SGF. By both the actions of adsorption and oxidation, AC may reduce the effect of loading and maintenance doses of NAC.