Blood collection from conscious, unanesthetized mice is often performed during the drug development process. The site of collection may influence the parameter(s) of interest. To investigate the potential influence of collection site on plasma glucose and insulin, a study was conducted to compare plasma glucose and insulin concentrations in blood samples collected without anesthesia from the retroorbital sinus versus the tail vein in 10- to 12-week-old female C57BL/6 mice. Two experiments were performed. In the first experiment, mice were randomized to be bled from the tail vein then the retroorbital sinus or vice-versa in a balanced two-period crossover design. In this experiment, the retroorbital and tail vein bleeds were performed a few minutes apart. The second experiment was similar to the first, except the bleeds were performed 1 week apart. Overall, retroorbital collection yielded lower glucose levels (p, 0.001) and higher insulin levels (p, 0.001) than did tail vein collection. The minimum difference in measured glucose in a retroorbital collection versus a tail vein collection, after adjusting for the effect of sequential bleeding was -97 mg/dl; the maximum was 98 mg/dl. We estimate that about 98% of observations taken under similar conditions would fall in this interval. The minimum difference in measured insulin in a retroorbital collection versus a tail vein collection was -0.6 ng/ml; the maximum was 7.3 ng/ml. We estimate that about 98% of observations taken under similar conditions would fall in this interval.