This study aimed to evaluate the accuracy and reliability of a new smartphone-based acoustic uroflowmetry compared to conventional uroflowmetry. Methods: This was a prospective validation study enrolling 128 subjects from September 2017 to April 2018 comparing a novel acoustic uroflowmetry to conventional uroflowmetry in an outpatient urologic clinic at Seoul National University Bundang Hospital. Visual comparison of flow patterns and uroflow parameters such as maximum flow rate (Qmax), average flow rate (Qavg), and voided volume were compared between the 2 techniques. Reliability and accuracy of the uroflowmetry results were compared using Pearson correlation coefficient (PCC) and Student t-test, respectively. Results: One hundred twelve participants were included in the study. Of these, 77 had baseline urologic comorbidities while 35 were normal participants. Flow patterns between the 2 uroflowmetry techniques demonstrated strong visual correlation. When compared to conventional uroflowmetry, all 3 parameters of voiding in male participants showed a very robust correlation with PCC of 0.88, 0.91, and 0.95 for Qmax, Qavg, and voided volume, respectively. Among female participants, we observed a PCC of 0.78, 0.93, and 0.96 for Qmax, Qavg, and voided volume, respectively. The Qmax showed a statistically significant difference in both sexes between the 2 methods, although the absolute value was small. Conclusions: Uroflowmetry using acoustic analysis demonstrates comparable findings to conventional uroflowmetry. This provides an opportunity to perform uroflowmetry in the clinic or at home in a reliable, inexpensive manner. Future large-scale prospective studies are required to further validate our results.