Degradation products of collagen type I can be measured by CrossLaps (CTX) immunoassays, providing an index of bone resorption. The CTX epitope EKAHDGGR comprises a DG-motif susceptible to post-translational modifications. In newly synthesized collagen this motif is in the native form denoted alphaCTX, but converts to an isomerized form (betaCTX) during aging of bone. Furthermore, the lysine residue (K) within the CTX epitope participates in inter-molecular cross-links in mature bone. The present paper describes an assay, ALPHA CTX ELISA for measurement of cross-linked alphaCTX molecules (alpha-alphaCTX) in urine. The ALPHA CTX ELISA demonstrated a high specificity and technical precision for measuring such fragments. The assay was evaluated in a cross-sectional study, comparing the urinary excretion of the marker in 100 breast cancer patients with bone metastases (BC+) and 15 breast cancer patients without metastases to bone (BC-) as well as 31 age-matched healthy post-menopausal women (PM). For comparison alpha, beta, and beta-betaCTX was also measured using commercially available immunoassays. In BC+ urinary alpha-alphaCTX increased significantly compared to BC- and PM with p-values of 0.005 and <0.0001, respectively. In contrast, the age-modified form beta-betaCTX, representing the degradation of old bone, was less increased. Z-score analysis was used to compare the ability of the CTX markers to discriminate between BC+ and PM. The alpha-alphaCTX marker was found to provide a far better discrimination (Z=7.5) than beta-betaCTX (Z=3.6). In conclusion, measurement of alpha-alphaCTX fragments may provide a clinically relevant assessment of bone resorption related to bone metastases.