C-Jun N-terminal kinase (JNK) activation has been shown to play a crucial role in the development of various types of cancer. IQ-1S is a JNK inhibitor based on the 11H-indeno[1,2-b]quinoxalin-11-one scaffold. The aim of this study was to investigate the antiproliferative effect of IQ-1S on MCF7 breast cancer cells in both two-dimensional (2D) monolayer and 3D multicellular spheroid test-systems. Non-adherent, non-tethered 3D objects were generated from single MCF7 breast cancer cells in a hydrogel array. IQ-1S was added directly to the cells seeded in the hydrogel array. MCF7 spheroids were grown for 7 days. Spheroid size, growth rate, and morphology were assessed at single-object resolution. The study revealed significant differences in the size, morphology and some vital characteristics of breast cancer 3D objects when treated with the JNK inhibitor compared to vehicle (dimethyl sulfoxide)-treated controls. Spheroids treated with IQ-1S (20 μM) after 7 days are significantly smaller than the control objects. This difference was not attributable to variations in the initial number of cells seeding for the spheroid formation. Morphological examinations showed that 3D multicellular objects grown from IQ-1S-treated cells lose their regular, round morphology, in contrast to control spheroids. Furthermore, cell proliferation measured using a label-free impedance monitoring platform was reduced in monolayer (2D) culture of MCF7 cells in the presence of 10 and 20 μM IQ-1S. MCF7 cells in 2D culture treated with IQ-1S (20 μM) for 72 and 153 h showed a significant increase in apoptosis as assessed by flow cytometry with annexin V/propidium iodide staining. An in silico evaluation showed that compound IQ-1S has generally satisfactory ADME (absorption, distribution, metabolism, and excretion) properties and high bioavailability. We conclude that IQ-1S effectively inhibits the growth of 3D spheroids and MCF7 cells in 2D culture and has a high potential for use in preclinical tumor growth models.