Reptiles are handled during transport, veterinary care, education programs, and as companion animals. This study investigated corticosterone levels (CS), heterophil/lymphocyte ratio (H/L), and behavioral responses to routine handling in the ball python (Python regius) (n=4) and the blue-tongued skink (Tiliqua scincoides) (n=3). We validated the RSL I125 Corticosterone Kit for rodents for parallelism and recovery, and found this to be a precise and highly specific means of CS measurement in reptiles. To determine the optimal blood sampling time, animals were sampled throughout a 24-hour cycle under two different light regimens, one a 12L:12D and one in which a red light was on during the dark period. The diurnal pattern of CS did not differ as a result of light regimen. Python peak CS levels occurred at 2400 hours during the peak locomotor activity period and at 1200 hours when body temperature was highest, while skinks showed no significant CS rhythm. Heterophil/lymphocyte ratio values indicated that cardiac puncture did not cause chronic stress although it may be an acute stressor. Animals were then handled gently, manually, or were container restrained (CR) for 10 minutes. Although skink CS was unaffected by treatment, CR caused an elevated level of CS in pythons which may indicate short-term stress. There were no differences in H/L ratio or changes in activity level in either species. Brief periods of routine handling of the study species in captivity thus did not cause chronic stress as measured by CS, H/L ratio, and activity parameters.