A rapid and sensitive method was established for arsenic (As) speciation based on high performance liquid chromatography coupled to inductively coupled plasma mass spectrometry (HPLC-ICP-MS). This method was validated for the quantification of four arsenic species, including arsenite (As^III), arsenate (As^V), monomethylarsonic acid (MMA^V) and dimethylarsinic acid (DMA^V) in cynomolgus macaque plasma. Separation was achieved in just 3.7 min with an alkyl reverse phase column and highly aqueous mobile phase containing 20 mM citric acid and 5 mM sodium hexanesulfonate (pH = 4.3). The calibration curves were linear over the range of 5–500 ng·mL⁻¹ (measured as As), with r > 0.99. The above method was validated for selectivity, precision, accuracy, matrix effect, recovery, carryover effect and stability, and applied in a comparative pharmacokinetic study of arsenic species in cynomolgus macaque samples following intravenous and intragastrical administration of arsenic trioxide solution (0.80 mg·kg⁻¹; 0.61 mg·kg⁻¹ of arsenic); in addition, the absolute oral bioavailability of the active ingredient As^III of arsenic trioxide in cynomolgus macaque samples was derived as 60.9 ± 16.1%.