Background: Chronic skin wounds are a common complication of many diseases such as diabetes. Various traditional methods for assessing skin wound closure are used in animal studies, including wound tracing, calliper measurements and histological analysis. However, these methods have poorly defined wound closure or practical limitations.Digital image analysis of wounds is an increasingly popular, accessible alternative, but it is unclear whether digital assessment is consistent with traditional methods. This study aimed to optimise and compare digital wound closure assessment with traditional methods, using a diabetic mouse model. Diabetes was induced in male C57BL/6J mice by high-fat diet feeding combined with low dose (65 mg/kg of body weight) streptozotocin injections. Mice fed normal chow were included as controls. After 18 weeks, four circular full-thickness dorsal skin wounds of 4 mm diameter were created per mouse. The wounds were photographed and measured by callipers. Wound closure rate (WCR) was digitally assessed by two reporters using two methods: wound outline (WCR-O) and re-epithelialisation (WCR-E).Wounded skin tissues were collected at 10-days post-wounding and wound width was measured from haematoxylin and eosin-stained skin tissue. Results: Between reporters, WCR-O was more consistent than WCR-E, and WCR-O correlated with calliper measurements. Histological analysis supported digital assessments, especially WCR-E, when wounds were histologically closed. Conclusions WCR-O could replace calliper measurements to measure skin wound closure, but WCR-E assessment requires further refinement. Small animal studies of skin wound healing can greatly benefit from standardised definitions of wound closure and more consistent digital assessment protocols.