This article describes a method for fabricating three-dimensional (3D) microfluidic devices using layered paper and double-sided adhesive tape. These devices, called 3D microfluidic paper analytical devices (μPADs), offer capabilities in microfluidics that are difficult to achieve with conventional open-channel microsystems made from glass or polymers. The 3D paper-based devices can wick fluids and distribute microliter volumes of samples from a single inlet point into arrays of detection zones, with numbers up to thousands. This capability allows for the execution of new analytical protocols simply and inexpensively on a piece of paper without external pumps. The devices are fabricated by stacking alternating layers of patterned paper and double-sided adhesive tape. The paper is patterned with hydrophilic channels and hydrophobic walls, while the tape is patterned with holes that connect channels in different layers of paper. This allows for complex microfluidic paths and significantly expands the capabilities of low-cost analytical systems. The devices are small, lightweight, and easy to stack, store, and transport, making them suitable for use in distributed healthcare in the developing world, environmental monitoring, and other applications requiring low cost, simplicity, and ruggedness. The μPADs distribute fluids both vertically and laterally, enabling streams of fluid to cross one another without mixing. They use capillary wicking to distribute fluids into complex arrays of detection zones in seconds to minutes. The devices can be used for diagnostics, drug development, and environmental monitoring. The article also describes the fabrication process, including patterning paper with SU-8 2010 photoresist and patterning tape with a laser cutter. The devices are tested with various samples and reagents, demonstrating their ability to perform assays and generate calibration curves. The combination of camera phones and μPADs provides a complete system for quantitative detection of analytes in resource-limited settings. The study concludes that the combination of paper, tape, and stacking makes 3D paper-based microfluidics practical for use in resource-limited and difficult environments, offering new functions and capabilities to microfluidic systems.This article describes a method for fabricating three-dimensional (3D) microfluidic devices using layered paper and double-sided adhesive tape. These devices, called 3D microfluidic paper analytical devices (μPADs), offer capabilities in microfluidics that are difficult to achieve with conventional open-channel microsystems made from glass or polymers. The 3D paper-based devices can wick fluids and distribute microliter volumes of samples from a single inlet point into arrays of detection zones, with numbers up to thousands. This capability allows for the execution of new analytical protocols simply and inexpensively on a piece of paper without external pumps. The devices are fabricated by stacking alternating layers of patterned paper and double-sided adhesive tape. The paper is patterned with hydrophilic channels and hydrophobic walls, while the tape is patterned with holes that connect channels in different layers of paper. This allows for complex microfluidic paths and significantly expands the capabilities of low-cost analytical systems. The devices are small, lightweight, and easy to stack, store, and transport, making them suitable for use in distributed healthcare in the developing world, environmental monitoring, and other applications requiring low cost, simplicity, and ruggedness. The μPADs distribute fluids both vertically and laterally, enabling streams of fluid to cross one another without mixing. They use capillary wicking to distribute fluids into complex arrays of detection zones in seconds to minutes. The devices can be used for diagnostics, drug development, and environmental monitoring. The article also describes the fabrication process, including patterning paper with SU-8 2010 photoresist and patterning tape with a laser cutter. The devices are tested with various samples and reagents, demonstrating their ability to perform assays and generate calibration curves. The combination of camera phones and μPADs provides a complete system for quantitative detection of analytes in resource-limited settings. The study concludes that the combination of paper, tape, and stacking makes 3D paper-based microfluidics practical for use in resource-limited and difficult environments, offering new functions and capabilities to microfluidic systems.