Endocrine therapy resistance in breast cancer is a major obstacle in the treatment of patients with estrogen receptor-positive (ER+) tumors. Herein, we demonstrate the feasibility of longitudinal, noninvasive and semiquantitative in vivo molecular imaging of resistance to three endocrine therapies by using an inducible fluorescence-labeled short hairpin RNA (shRNA) system in orthotopic mice xenograft tumors. We employed a dual fluorescent doxycycline (Dox)-regulated lentiviral inducer system to transfect ER+ MCF7L breast cancer cells, with green fluorescent protein (GFP) expression as a marker of transfection and red fluorescent protein (RFP) expression as a surrogate marker of Dox-induced tumor suppressor phosphatase and tensin homolog deleted on chromosome 10 (PTEN) knockdown. Xenografted MCF7L tumor-bearing nude mice were randomized to therapies comprising estrogen deprivation, tamoxifen or an ER degrader (fulvestrant) and an estrogen-treated control group. Longitudinal imaging was performed by a home-built multispectral imaging system based on a cooled image intensified charge coupled device camera. The GFP signal, which corresponds to number of viable tumor cells, exhibited excellent correlation to caliper-measured tumor size (P