Blood examination is a key element in studies of laboratory animals. In rodents, retrobulbar venous plexus puncture is a commonly used method for obtaining a blood sample. Although this technique yields large volumes of blood, the disadvantage is that it can lead to severe tissue damage. The aim of the present study was to develop the puncture of V. sublingualis as a suitable alternative technique for drawing blood in mice and other rodents. In rats, this method has been established for collecting large blood volumes. During the first part of the study, the sublingual bleeding technique was developed for use in mice and hamsters. Guinea pigs, however, do not have a sublingual vein; therefore, in this species the method is not possible. In the second part of the study, retrobulbar and sublingual methods were compared using male CD-1 mice. When compared with the retrobulbar method, sublingual venepuncture showed less tissue destruction in mice, with a decreased mean severity in the histological examination. In conclusion, sublingual venepuncture can be recommended as a suitable, alternative blood collection technique, because of the reduced risk of tissue damage in mice and hamsters.