The use of serum alkaline phosphatase (ALP) isoenzymes as markers of breast cancer metastases and treatment efficacy has received little attention. Twenty-six breast cancer women (56+/-13 years, all post-menopausal) were prospectively evaluated during their first and third course of chemotherapy (4-week interval). Serum samples were analyzed for ALP isoenzymes (bone, liver, and intestine) using a lectin affinity electrophoresis kit (Hydragel 15 ISO-PAL, Sebia) adapted on a semi-automated Hydrasys system (Sebia). Results were compared with imaging techniques for the presence of metastases; bone ALP isoenzyme (B-ALP) results were compared with C-Terminal degradation products of type I collagen (S-CTX) (CrossLaps, IDS Nordic). Serum B-ALP, but not S-CTX, confirmed the presence of bone metastases (BM) (n=15) with 67/100% sensitivity/specificity (using a 69 UI/L ROC cut-off); ROC AUC was 0.806 (P=0.0004) (NS for S-CTX). Chemotherapy reduced serum B-ALP by 24% over 4 weeks (P=0.0012); there was no change for S-CTX. There was no specific clinical pattern for other ALP isoenzymes (liver and intestine). In conclusion, serum B-ALP, but not S-CTX, could help confirm the presence of BM in breast cancer patients.