Microfluidic 3D cell culture is a promising technology for the screening of drug toxicity profiles. In this study, a bioartificial liver consisting of a surfaceengineered microfluidic silicon chip with microtrenches mimicking hepatic sinusoids is shown to extend 3D primary hepatocyte culture and improve in vitro drug screening for hepatotoxicity, with respect to the state-of-the-art literature on this subject. Primary hepatocytes hosted in the 3D heparin-coated microtrenches (the bioartificial liver) secrete high levels of albumin and urea over 4 weeks. The cytotoxicity of common drugs, namely, acetaminophen, chlorpromazine, and tacrine, was assessed on primary hepatocytes both at day 1 and day 7. The results suggest that mimicking hepatic sinusoids using a microtrench format allows the maintenance of difficult-to-culture primary hepatocytes to be extended to 4 weeks and provides an alternative model to animal studies for the screening of the cytotoxicity of new drugs.