Cyclophosphamide (CPA) dosing by body surface area (BSA, m2) has been questioned as a predictor for individual drug exposure. This study investigated phosphoramide mustard-hemoglobin (PAM-Hb, pmol g-1 Hb) as a biomarker of CPA exposure in 135 female breast cancer patients receiving CPA during three courses based on BSA: 500 mg/m2 (C500 group, n = 67) or 600 mg/m2 (C600 group, n = 68). The inter-individual difference was calculated for both groups by dividing the highest through the lowest PAM-Hb value of each course. The inter-occasion difference was calculated in percentage for each individual by dividing their PAM-Hb value through the group mean per course, and subsequently dividing this ratio of the latter through the previous course. A multivariable linear regression (MLR) was performed to identify factors that explained the variation of PAM-Hb. During the three courses, the inter-individual difference changed from 3.5 to 2.1 and the inter-occasion difference ranged between 13.3% and 11.9% in the C500 group. In the C600 group, the inter-individual difference changed from 2.7 to 2.9 and the inter-occasion difference ranged between 14.1% and 11.7%. The MLR including BSA, age, GFR, and albumin explained 17.1% of the variation of PAM-Hb and was significantly better then the model including only BSA. These factors should be considered when calculating the first dose of CPA for breast cancer patients.