Theraphosid spiders have become increasingly popular for private and public uses in the United States. However, little is known about their physiology from a medical standpoint. This study represents the first attempt to establish reference hemolymph values for two common species of theraphosids, the goliath birdeater spider (Theraphosa blondi) and the Chilean rose spider (Grammostola rosea). Eleven T. blondi and twelve G. rosea, all wild-caught subadults, were obtained after importation and hemolymph was collected for biochemical analysis. After 8 wk of captivity, hemolymph was again collected from the spiders and analyzed. The biochemical analytes measured in the study included aspartate transferase, creatine kinase, glucose, total protein, albumin, uric acid, blood urea nitrogen, phosphorous, calcium, potassium, and sodium. The osmolality of the hemolymph was estimated for each spider using two different formulae. There were significant differences in body weight, sodium, potassium, and osmolality between the sampling times for both species. There were also significant differences in creatine kinase, calcium, total protein, and blood urea nitrogen between sampling periods for T. blondi. The results of this study suggest that serial hemolymph samples may be used to assess the hydration status of theraphosid spiders. In addition, the differences in hemolymph analytes between spiders suggest that there may be differences between species that should be addressed in future studies.