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This work presents, for the first time, a novel cross injection analysis (CIA) system for the simultaneous multi-determination of key biomarkers associated with diabetic nephropathy-namely, albumin, creatinine, and glucose-within a single analytical run. Unlike conventional flow-based techniques that rely on sequential measurements, the proposed CIA platform integrates multiple analytical pathways into a unified design, enabling one-shot multi-analyte analysis without the need for complex separation units or injection valves. The system employs peristaltic pumps and a rectangular platform with orthogonal flow channels, allowing concurrent aspiration and efficient transport of reaction products to compact detectors. Albumin determination was based on ion-association with tetrabromophenolphthalein ethyl ester. Creatinine was measured using the Jaffé reaction. Glucose was colorimetrically detected via its reaction with 3,5-dinitrosalicylic acid. The developed CIA provides enhanced sensitivity through its pre-mixing effect, enabling reliable quantification of trace analytes. Excellent analytical performance was achieved, including wide linear ranges (r² > 0.99), good precision (RSD < 7%), and rapid analysis (5 min). The method was validated against established reference methods, showing no significant differences, and successfully applied to urine with satisfactory recoveries (84.8-107.3%). Importantly, the proposed system adheres to green chemistry by minimizing reagent consumption and waste generation, offering a sustainable approach for multi-parameter clinical analysis.