Introduction. Regenerative medicine is a rapidly expanding discipline. However due to a lack of validated outcome measures, clinical trials have been far few. This study aims to assess the validity, inter-observer reliability and intra-observer reproducibility of experimental fracture healing assessment on plain radiographies. This technique involves implantation of mesenchymal stem cell (MSC) seeded constructs on only one side of the fracture after randomisation. Methods. We examined inter/intraobserver agreement on the area and “bridging length” of callus formed on opposite sides of the fracture. Among 16 orthopaedic surgeons with trauma commitments (8 consultants, 8 registrars) on two separate occasions (average 52 days apart). They independently assessed the radiographs (AP or lateral) of 28 patients with fractures of the tibial or femoral shaft. The fractures chosen included non-unions treated with MSC/constructs and fresh fractures at 4–9 months. For each radiograph the assessor assigned which side (medial or lateral) is there more callus. Chase-corrected agreement using Fleiss kappa was used to compare opinions. Digital analysis software (Image-J) was used to quantify extent/bridging callus and correlate it with surgeons opinion. Results. Inter-observer variation showed a substantial overall agreement (k = 0.716) on the fracture side containing a larger “area” of callus but moderate agreement (k = 0.489) on side with more “bridging length”. These results were reproducible with a substantial overall intraobserver agreement. MSC/construct treated non-union showed a larger amount of agreement than fresh fractures for area (k = 0.754 vs 0.613) and bridging (0.550 vs 0.406). Utilizing digital analysis, non-unions showed a significant larger quantifiable difference between sides than fresh fractures (p = 0.009) for area but not bridging length (p = 0.269). Digital analysis quantification and surgeons opinion showed an almost perfect agreement for area (k = 0.867) and bridging (k = 0.846). Discussion. In this study we aimed to validate a novel method at studying the efficacy and effect of regenerative techniques on fracture healing. In particular, plain radiographs for comparing a treatment/internal control side. In this study we showed this method assessing area of callus is valid, reliable and reproducible. This is particularly so for MSC/construct treated non-union where the difference in both sides is higher as quantified in digital analysis. This is a novel method of experimental fracture healing using an internal control which decreases the variation between groups and sample size needed. This makes regenerative medicine clinical trials easier