Collecting blood from laboratory animals is necessary for a wide variety of scientific studies, but the small size of the zebrafish makes this common procedure challenging. We developed a novel, minimally invasive method to collect repeated blood samples from adult zebrafish. This method minimizes trauma to the zebrafish and yields a low mortality rate of 2.3%. The maximum volume of blood that can be collected using this technique is approximately 2% of body weight. To avoid blood loss anemia and hemorrhagic death, we recommend that the total blood sample volume collected over repeat bleeds should be ≤0.4% of body weight per week, and ≤1% of body weight per 2 weeks. Additionally, we applied this method to the study of zebrafish glycolipid metabolism by measuring blood glucose and plasma triacylglyceride levels weekly over a 5-week period in both control and overfed zebrafish. The overfed fish developed significantly increased fasting blood glucose levels compared with normally fed fish. This new method of blood collection is essential for zebrafish or other small aquarium fish research requiring repeated blood samples, and increases the utility of the zebrafish as a model animal in hematological studies of human diseases.