The recent US FDA Modernization Act 2.0 has intensified interest in microfluidic-based in vitro systems for preclinical research. However, the high costs of conventional microfabrication methods and commercial devices present significant barriers, particularly in developing countries. This study addresses these challenges by exploring the use of industrial-grade acrylic sheets and a conventional CO2 laser for cost-effective microfabrication. The research investigates the effects of laser parameters—specifically nozzle speed and power—on the dimensions and quality of microchannels, surface roughness, and bonding strength. Results indicate that increased nozzle speed reduces kerf width and enhances microchannel quality, whereas high laser power (∼52 watts) induces bubbling at the edges. A simple acetone and ethanol mixture was found to provide optimal bonding strength without altering the acrylic’s molecular structure. UV spectrophotometry revealed minimal changes (∼6 %) in visible light transmittance due to bonding conditions, while FTIR analysis showed no residuals that could potentially cause cytotoxicity. Despite variations in surface roughness resulting from the etching process, cell viability remained unaffected. The surfaces supported effective cell culture for lung (A549), neuron (SH-SY5Y), and liver (HepG2) cell lines. This approach presents a practical and cost-effective solution for developing in vitro platforms, making it an accessible option for research and academic applications.