Tumor collagen characteristics influence tumor malignancy, invasion, and metastasis. This study investigates the effects of trastuzumab (Tz) on the collagen of Tz-responsive (BT474) and Tz-resistant (HR6) breast cancer xenografts. Collagen content was assessed by in vivo second harmonic generation (SHG) imaging and histological trichrome staining of tumor sections. Collagen SHG imaging of control BT474 and HR6 tumors demonstrated increased collagen density after 14 days of treatment (). Trichrome staining revealed decreased collagen in Tz-treated BT474 and HR6 tumors at 2, 5, and 14 days of treatment, suggesting that Tz affects the tumor microenvironment independent of epithelial cell response. Additionally, collagen alignment analysis revealed significantly less aligned collagen in the Tz-treated BT474 tumors at day 14 compared with control BT474 tumors. There was no correlation between SHG endpoints (collagen density and alignment) and trichrome staining (), consistent with the physically distinctive nature of these measurements. There was also no correlation between tumor size and collagen endpoints (). These results identify changes within the collagen compartment of the tumor microenvironment following Tz treatment, which are independent from the tumor cell response to Tz, and demonstrate that intravital collagen SHG imaging is capable of measuring dynamic changes in tumor microenvironment following treatment that complements trichrome staining.