Colistin is essential for treating multidrug-resistant Gram-negative bacterial infections but has significant nephrotoxic side effects. Traditional approaches for studying colistin's nephrotoxicity are challenged by the rapid metabolism of its prodrug, colistin methanesulfonate and the difficulty of obtaining adequate plasma from critically ill patients. To address these challenges, we developed the Spheroid Nephrotoxicity Assessing Platform (SNAP), a microfluidic device that efficiently detects colistin-induced toxicity in renal proximal tubular epithelial cell (RPTEC) spheroids within 48 hours using just 200 μL of patient plasma. Our findings demonstrate that SNAP not only promotes higher expression of kidney-specific markers aquaporin-1 (AQP1) and low-density lipoprotein receptor-related protein 2 (LRP2) compared to traditional two-dimensional (2D) cultures, but also exhibits increased sensitivity to colistin, with significant toxicity detected at concentrations of 50 μg ml-1 and above. Notably, SNAP's non-invasive method did not identify nephrotoxicity in plasma from healthy donors, thereby confirming its physiological relevance and showcasing superior sensitivity over 2D cultures, which yielded false-positive results. In clinical validation, SNAP accurately identified patients at risk of colistin-induced nephrotoxicity with 100% accuracy for both early and late onset and demonstrated a 75% accuracy rate in predicting the non-occurrence of nephrotoxicity. These results underline the potential of SNAP in personalized medicine, offering a non-invasive, precise and efficient tool for the assessment of antibiotic-induced nephrotoxicity, thus enhancing the safety and efficacy of treatments against resistant bacterial infections.