Arsenic (As) is a ubiquitous, toxic and carcinogenic element, and As compounds can pose significant health risks to human and animals. The toxicity of As depends on both the total concentrations and the chemical species as well. Therefore, much attention has been paid to As speciation analysis in the environmental and biological samples. Previous studies claimed that methanol-water solvent system is one of the most frequently utilized methods for extraction of arsenic in samples, and methanol can improve As sensitivity by ICP-MS. However, the effect of methanol addition on As speciation analysis is largely unknown. In this study, the potential As speciation changes in methanol-water solvent mixture covering the range of 0~100% were investigated. Moreover, an optimized condition was employed by using high-performance liquid chromatography inductively coupled plasma mass spectrometry (HPLC-ICP-MS) for simultaneous determination of six As species including AsC, AsB, As(Ⅲ), DMA, MMA and As(Ⅴ). In accordance with previous studies, the HPLC separation was performed on anion-exchange column using a mobile phase of 30 mmol·L-1 (NH4)2CO3 at pH=9.3. The ICP-MS on-line detection of As (AsO+, m/z=91) in eluting compounds was accomplished using the dynamic reaction cell (DRC) technology with oxygen as reaction gas. In addition, the method validation with optimized conditions indicated that the coefficients were above 0.998 9 for all As species. The results indicate that the peak area measured in the chromatographic signal for As(Ⅲ) and DMA increased significantly with the methanol contents, indicating significant impact of methanol contents on As speciation analysis. A test with comparison of 25% methanol solvent mixture, which showed the minimum effects on As speciation changes, and purified water, was further performed, and the results showed that As(Ⅲ) and DMA concentration increased markedly compared to those diluted by purified water but little change for the other four As species. Thus, the elimination of methanol plays an important role in As speciation analysis.