Tarantulas are a commonly kept species that are occasionally presented to veterinarians in exotic practice. A recent study on Grammastola rosea hemolymph biochemistry has been performed with nonanesthetized adult theraphosids. The objective of this study was to produce reference intervals for biochemistry biomarkers in hemolymph of chemically restrained G. rosea for use diagnostically by exotic veterinarians. Cardiac hemolymph collection was performed on 20 subadult tarantulas under general anesthesia with isoflurane. Samples were processed by a commercial laboratory. Statistics performed on the data include outlier exclusion, descriptive statistics, normality tests, and Pearson correlations. Reference intervals were made for total protein, creatine kinase (CK), aspartate aminotransferase (AST), glucose, uric acid (UA), calcium, and phosphorus. No cortisol was detected. The majority of the intervals produced were normally distributed with the exceptions of UA, phosphorus, and CK. Pearson correlation tests found several significant (P = <0.05) correlations between variables. The majority of the data displayed a normal distribution, unlike the previous study, with a greater number of replicates. The total protein, glucose, UA, calcium, and AST values generated were similar to those reported in the previous study. Conversely several variables such as phosphorus, CK, and albumin were not consistent with those previously reported. Evidence is presented for a lack of albumin, CK, and AST in Arachnida and thus previous data for these proteins is likely to be artifactual.