Electrochemical biosensors have the potential to transform biomarker analysis by replacing bulky laboratory equipment with portable devices. This transition would allow routine clinical tests to be performed in primary care settings, facilitating early disease detection and timely intervention. Over the past decade, activities have shifted towards fully-integrated microsystems, with increasing research efforts dedicated to developing low-cost, disposable devices made from accessible materials, such as plastic or paper. Unfortunately, the fabrication of the reference electrode in these low-cost electrochemical cells remains a significant and unmet challenge. A typical electrochemical detector includes a reference electrode that acts as a reference potential for the working electrode. To ensure measurement stability, the reference electrode is usually made by a standard material with a known redox potential, such as silver/silver chloride or the saturated calomel electrode. However, these materials are often incompatible with common microfabrication techniques or demand complex fabrication processes, thus hindering their integration into low-cost disposable cartridges. Circumventing the overreliance on reference electrodes would enable researchers to design electrochemical sensors using a far broader array of substrates, and thus pave the way towards simpler and more accessible diagnostic technologies. A popular approach is to use an uncoated electrode material, such as carbon or gold, as the reference electrode (i.e. pseudo-reference electrodes). However, because the electric potential of pseudo-reference electrodes strongly depends on their chemical environment, this approach fails to deliver a consistent reference potential in continuous flow detection assays. As a result, the integration of dedicated silver/silver chloride electrodes remains the norm in flow injection analysis (FIA), hindering the fabrication of low-cost disposable devices. FIA is a widely used analytical technique that enables reliable and high-throughput testing of multiple samples by injecting them into a continuous flow; its rapid and automated nature makes it particularly attractive for clinical chemistry. In the realm of point-of-need testing, paper-based FIA devices have attracted considerable attention due to low fabrication costs and their passive, pump-free operation. Because of the difficulty of fabricating electrodes in cellulose paper, early attempts to incorporate electrochemical sensing into paper-based FIA relied on the use of external electrodes in contact with the fluidic path. Such a strategy essentially undermines the advantage of paper as a single-use substrate, since the external electrodes are reused multiple times, raising the risk of crosscontamination. To address this shortcoming, we recently developed laser-pyrolysis of cellulose as a versatile technology for fabricating electrodes fully embedded into paper. Despite their advantages, the electrochemical sensors fabricated in this manner still relied on a three-electrode configuration, with a laser-induced graphenized (LIG) pseudo reference, resulting in signal aberration in continuous-flow assays. The adoption of two-electrode, or reference-free, electrochemical sensors, has the potential to address many of the challenges associated with the use of pseudo-references in FIA. Moving to two-electrode systems would substantially simplify device fabrication and readout hardware by eliminating the need for a reference potential altogether. Unfortunately, due to their unconventional nature and our limited insight of their workings, two-electrode systems remain underexplored and are often used empirically without rational design. Accordingly, understanding the inherent limitations of these systems is essential to harnessing their full potential and ensuring widespread adoption. Here, using paper-based laser-pyrolyzed devices as a model system, we demonstrate that two-electrode detection can provide excellent performance in FIA cartridges for clinical analysis. As proof of concept, we designed a cartridge to perform urinary creatinine analysis, an essential diagnostic assay in need of miniaturization and automation. Urinary creatinine serves as a key biomarker for kidney function, as its production is steady and proportional to a healthy individual's muscle mass. It is also widely used as a normalizing factor in urine assays, such as the albumin-to-creatinine ratio, or as an internal control for sample collection adequacy. The gold-standard method for quantifying urine creatinine remains the Jaffe reaction, which is a colorimetric reaction between creatinine and picric acid developed in 1886 (Fig. ). However, this assay has limitations, e.g. cross-reaction with glucose, and is not suitable for miniaturization and high-throughput analysis due to the need of bulky and expensive optics for signal readout. A number of electrochemical approaches for creatinine analysis have been proposed, including those based on iron ion mediators, picric acid detection, or direct detection catalyzed by copper nanoparticles. Unfortunately, these approaches suffer from slow electron transfer, large redox potential or demanding tedious electrode modification, respectively. Thus, they are unsuitable for use in low-cost disposable devices.