A method for blood collection from the jugular vein of mice without anesthesia was compared with a tail-incision technique. Jugular vein blood collection allowed withdrawal of almost 15% of the circulating blood volume at a time in less than 1 min. Hemolysis, hematocrit, and plasma thrombin-antithrombin complexes (a marker of blood coagulation) were higher in samples collected from the tail vein than the jugular vein. Mice produced similar plasma corticosterone levels after serial blood collection by either method. Tail incision led to a slight but significant increase in C-reactive protein levels. Using the jugular venipuncture technique, we then performed a pharmacokinetic study and an oral glucose tolerance test. Plasma concentrations of levofloxacin, an antimicrobial agent, were dose-dependently elevated after oral administration, and linear increases in C(max) and AUC were observed. We also confirmed that overall glucose excursion is significantly decreased in mice treated with exendin 4, a glucagon-like peptide 1 agonist. These results indicate that the jugular venipuncture is a useful technique from the point of view of no requirement for anesthetics, serial blood collection at short intervals, large volume of blood collection, quality of sample and animal welfare. This technique is of particular interest for studies that examine time-dependent changes in blood variables.