Urinary stone treatment has been strongly influenced by advances in technology. Nevertheless, the photonic characteristics of stones as the treatment target have been neglected. Monitoring fluorescence spectra is sufficient for automatic target differentiation and laser feedback control as previously described. We investigated the characteristics of fluorescence signals and the clinical practicability of real-time laser feedback control during lithotripsy. Fluorescence excitation light was superimposed on a holmium laser beam into the treatment fiber. Spectra were recorded and signal amplitude changes were analyzed during increases in distance between the fiber tip and the stone to identify the optimal threshold level for stone recognition. Ho:YAG lithotripsy was performed under in vitro surgical conditions in porcine tissue while our feedback system autonomously controlled the laser impulse release during lithotripsy. The tissue was then endoscopically and macroscopically examined for laser induced lesions. Mean ± SD autofluorescence signal amplitudes from urinary stone samples varied between 142 ± 29 and 1,521 ± 152 ADU while tissue and endoscope coating emission was negligible. Signal amplitude decreased rapidly at distances larger than 1 to 2 mm. Clinically reliable threshold values for target recognition could be set to prevent laser pulse emission if the stone was out of range or urothelial tissue might be harmed by laser irradiation. We observed no incorrectly released laser pulse or injury to tissue during autonomously controlled holmium laser lithotripsy. Our laboratory study strengthens the evidence that tracking real-time autofluorescence spectra during endoscopic stone surgery via automatic feedback control of the laser impulse release may become a potentially useful clinical tool for surgeons who navigate in the upper urinary tract.