A microfluidic biosensor paper-based analytical device, or μPAD, was developed as a point-of-care assay platform for the simultaneous determination of three clinically important markers: uric acid, urea, and triglycerides. To achieve a simultaneous determination, thermosensitive valves based on N-isopropylacrylamide polymers controlled the flow of fluid in the μPAD. The evaluation of the analytical characteristics of the μPAD was done using a photographic camera at room temperature.The detection limits were 4.5·10−5, 2.5·10−3, and 1.5·10−3 mg mL−1, for uric acid, urea, and triglycerides, respectively, and the precision expressed as relative standard deviation (% RSD) 3.2, 1.2, and 1.6%. The obtained μPAD devices were validated with real human plasma samples, demonstrating high accuracy and precision. Multiple analyte point-of-care tests such as those developed here have excellent characteristics, being easy to manufacture, cost-effective, easy to use, and highly sensitive.