Lab-on-a-chip technologies have the potential to deliver significant technological advances in modern biomedicine, through the ability to provide appropriate low-cost microenvironments for screening cells. However, to date, few studies have investigated the suitability of poly(dimethylsiloxane) (PDMS) for live cell culture. Here, we describe an inexpensive method for production of reusable, optical-grade PDMS microculture chips which provide a static and self-contained microwell system analogous to conventional polystyrene multiwell plates. We use these structures to probe the effects of PDMS upon live cell culture bioassays, using time-lapse fluorescence imaging to explore the toxicity of the substrate. We use three model systems to explore the efficacy of the microstructured devices: (i) live cell culture, (ii) adenoviral gene delivery to mammalian cells, and (iii) gravity enforced formation of multicellular tumor spheroids (MCTS). Results show that PDMS is nontoxic to cells, as their viability and growth characteristic in PDMS-based platforms is comparable to that of their polystyrene counterparts.