<p>Patients with critical illness often exhibit profound biological heterogeneity, complicating the identification of effective interventions. Resolving distinct molecular profiles to enable timely treatment decisions remains challenging, as integrating biomarker-guided care into routine monitoring is often hindered by fragmented, batch-based workflows. These manual operations decouple molecular data from the acute clinical timeline and are a barrier to reliable, real-time, near to patient, multi-center implementation. To address this gap, we developed an integrated microfluidic digital immunoassay system that achieves high analytical fidelity through a fully automated, simple workflow. The system utilizes a monolithic disposable cartridge to automate bead-based analyte capture, oil-phase partitioning, and signal amplification, eliminating the manual handling and emulsion steps that typically compromise digital assay robustness. The platform enables protein measurement within 45 minutes, achieving sub-picogram limit of detection ( &lt; 0.13 pg/mL), a dynamic range spanning three orders of magnitude, and strong analytical reproducibility. We demonstrate the clinical utility of the system by profiling a validated panel of inflammatory biomarkers in plasma from critically ill pediatric patients, using low sample volumes. Results show strong agreement with gold-standard multiplex assays (R² = 0.925-0.979). By providing a scalable framework for high-fidelity molecular profiling, this system supports the broader goal of accessible, multi-center biomarker validation and the practical implementation of precision medicine in critical care.</p><p></p>