Traditional two-dimensional (2D) cell culture models are limited in their ability to reproduce human structures and functions. On the contrary, three-dimensional (3D) microtissues have the potential to permit the development of new cell-based assays as advanced in vitro models to test new drugs. Here, we report the use of a dehydrated gelatin film to promote tumor cells aggregation and 3D microtissue formation. The simple and stable gelatin coating represents an alternative to conventional and expensive materials like type I collagen, hyaluronic acid, or matrigel. The gelatin coating is biocompatible with several culture formats including microfluidic chips, as well as standard micro-well plates. It also enables long-term 3D cell culture and in situ monitoring of live/dead assays.