Optimally preserved urinary exfoliated renal proximal tubule cells were assessed by multispectral imaging of cell autofluorescence. We demonstrated different multispectral autofluorescence signals in such cells extracted from the urine of patients with healthy or diseased kidneys. Using up to 10 features, we were able to differentiate cells from individuals with heathy kidneys and impaired renal function (indicated by estimated glomerular filtration rate (eGFR) values) with the receiver operating characteristic area under the curve (AUC) of 0.99. Using the same method, we were also able to discriminate such urine cells from patients with and without renal fibrosis on biopsy, where significant differences in multispectral autofluorescence signals (AUC = 0.90) were demonstrated between healthy and diseased patients (p < 0.05). These findings show that multispectral assessment of the cell autofluorescence in urine exfoliated proximal tubule kidney cells has the potential to be developed as a sensitive, non-invasive diagnostic method for CKD.