Objectives: To design novel 3D in vitro co-culture models based on the RGD-peptide-induced cell self-assembly technique. Results: Multicellular spheroids from M-3 murine melanoma cells and L-929 murine fibroblasts were obtained directly from monolayer culture by addition of culture medium containing cyclic RGD-peptide. To reach reproducible architecture of co-culture spheroids, two novel 3D in vitro models with well pronounced core–shell structure from tumor spheroids and single mouse fibroblasts were developed based on this approach. The first was a combination of a RGD-peptide platform with the liquid overlay technique with further co-cultivation for 1–2 days. The second allowed co-culture spheroids to generate within polyelectrolyte microcapsules by cultivation for 2 weeks. M-3 cells (a core) and L-929 fibroblasts (a shell) were easily distinguished by confocal microscopy due to cell staining with DiO and DiI dyes, respectively. Conclusions: The 3D co-culture spheroids are proposed as a tool in tumor biology to study cell–cell interactions as well as for testing novel anticancer drugs and drug delivery vehicles.