External apical root resorption is a common, yet unexplained, phenomenon associated with orthodontic treatment. Available methods of clinical evaluation are radiographic. Biochemical assays offer the advantage of being non-invasive, as well as being diagnostic and potentially prognostic. The hypotheses are firstly that during the process of root resorption, organic matrix proteins are released into the gingival crevicular fluid (GCF) and, secondly, that there is a difference in the levels of these proteins between a group of patients with mild root resorption and a control group. GCF was collected from the permanent central incisors of untreated subjects (controls, n = 20), primary second molars with half of the root resorbed (primary group and positive controls, n = 20) and permanent central incisors with mild root resorption in patients undergoing active orthodontic treatment (orthodontic group, n = 20). Dentine phosphoproteins (DPP) were measured in the GCF using an enzyme-linked immunosorbent assay developed with DPP isolated from human first premolars and an antibody against rat incisor DPP. The primary group showed the highest levels of DPP in the GCF compared with the orthodontic (P = 0.296) and control (P = 0.001) groups. The orthodontic group showed elevated levels relative to the control group (P = 0.046). It is concluded that root resorption can be studied using a biochemical immunoassay and that this method can provide quantitative measurement of DPP in GCF.