Triple-negative breast cancer (TNBC) accounts for 15-20% of breast cancer. There is limited effective targeted therapeutics for TNBC, and non-specific chemotherapy is the main method of treatment, which leads to various damaging side effects. In many TNBC cases, the disease often reoccurs or progresses after an initial successful response, with future resistance to therapy. A major contributor to this survival of disease is breast cancer associated fibroblasts (BCAFs), which signal to the tumour cells and their microenvironment to facilitate tumour growth, progression, invasion, and metastasis. Hence, TNBC is considered an unaddressed medical condition that urgently needs novel effective targeted therapeutics. To develop new therapeutics, we need three-dimensional preclinical models that can recapitulate the spatial dimension, cellular heterogeneity, and molecular networks of the tumour microenvironment more adequately than the conventional cell models. Aims: The aims are to develop a TNBC tumour spheroid model and determine its suitability for assessing the efficacy of novel drug treatments. Methods: TNBC and BCAF spheroids were generated, separately and as co-cultures, using low attachment 96-well plates, with liquid overlay. Response to a novel peptide-drug conjugate and nanoparticles, was assessed in spheroids by spheroid morphology assessment and cell viability assay. Uptake of compounds was confirmed using fluorescent microscopy (Incucyte S3). Results: We have successfully developed different types of spheroids. We demonstrated that the novel anticancer treatment showed high penetration to the spheroid structure leading to their complete disintegration and reduced viability. Significance: This project could lead to the development of a more clinically relevant preclinical model of TNBC for the development of potent and selective anticancer pharmaceuticals.