A liquid chromatography–tandem mass spectrometry (LC–MS/MS) method was developed for simultaneous determination of 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside (AICA riboside) and its active metabolite 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranotide (AICA ribotide) in mice plasma. Adenosine monophosphate was chosen as an internal standard, and plasma was deproteinized by adding 14% perchloric acid, and then, the resulting supernatant was diluted with water to decrease matrix effect. A 10 μL aliquot of the solution was injected into the LC–MS/MS system, and separation was achieved on a Gemini C6-Phenyl column with a mobile phase composed of 0.1% formic acid (v/v) and methanol. A triple quadrupole mass spectrometer with an electrospray turbo ion source operating in multiple reaction monitoring (MRM) positive ion mode was used as detector. The precursor–product ion pairs for MRM were m/z 259.2 → 110.1 and 259.2 → 242.1 for AICA riboside, 339.1 → 110.1 and 339.1 → 322.1 for AICA ribotide, and 348.3 → 136.0 and 348.3 → 119.1 for the internal standard, respectively. The total analytical run time was 10 min. The proposed method was linear over the range 0.01–10 μg/mL for AICA riboside and 0.01–5 μg/mL for AICA ribotide. The lower limit of quantification (LLOQ) was 10 ng/mL for both AICA riboside and AICA ribotide. The sensitivity, accuracy, and precision of this method were within acceptable limits during the validation period. The method was successfully applied to investigate the pharmacokinetics characteristics of AICA riboside and AICA ribotide in mice.