Different animals and biomarkers have been used to measure arsenic (As) relative bioavailability (RBA) in contaminated soils. However, there is a lack of comparison of As-RBA based on different animals (i.e., swine and mouse) and biomarkers [area under the blood As concentration curve (AUC) after a single gavaged dose vs. steady state As urinary excretion (SSUE) and As accumulation in liver and kidneys after multiple doses via diet]. In this study, As-RBA in 12 As-contaminated soils with known As-RBA via swine blood AUC model were measured using mouse blood AUC, SSUE, liver and kidneys analyses. Arsenic-RBA for the four mouse assays ranged 2.8–61%, 3.6–64%, 3.9–74%, and 3.4–61%, respectively. Compared to swine blood AUC assay (7.0–81%), though well correlated (R2 = 0.83), the mouse blood AUC assay generally yielded lower values. Similarly, strong correlations were observed between mouse blood AUC and mouse SSUE (R2 =0.86) and between urine, liver, and kidneys (R2 = 0.75–0.89), suggesting As-RBA values were congruent among different animals and endpoints. Selection of animals and biomarkers would not significantly influence the role of in vivo assays to validate in vitro assays. Based on its simplicity, mouse liver or kidney assay following repeated doses of soil-amended diet is recommended for future As-RBA studies.