Repeated blood sampling from laboratory animals is desirable in certain experimental designs and also for reducing the number of animals used in research. Biochemical methods for analysing blood samples require only small blood volumes to be collected (typically 20–40 µL). In juvenile mice, the small blood volume of the animals also requires only small samples to be taken. Furthermore, for behavioural studies it is desirable to have a method that does not require anaesthesia or the use of invasive indwelling cannulae. We report the validation of a refined method for repeated blood sampling (up to 3 times at 24 h intervals) in juvenile and adult mice using the tail incision method to sample from the lateral tail vein. This method is not stressful, as assessed by low basal levels of the stress hormone corticosterone. Since repeated blood samples can be collected from the same animal at multiple time points, it is not necessary to increase group size for terminal sample collection. Thus, in addition to being a refined method requiring no warming of the tail, no anaesthesia and only gentle restraint, this method also reduces the numbers of mice used for experiments.