This study was performed to compare the levels of pain experienced by young infants undergoing either suprapubic aspiration (SPA) or transurethral catheterization (TUC) for the collection of sterile urine samples. This prospective randomized clinical trial was conducted in hospitalized neonates in a university-affiliated hospital. Patients who required urine cultures were randomly assigned into one of two groups, the SPA or TUC group. The infants' faces were videotaped, and the changes in the facial expression and physiological parameters during the procedure were scored using the Premature Infant Pain Profile (PIPP) in a blind manner. The primary outcome was the severity of the pain experienced during each procedure, and the secondary outcomes were the success rate, the duration, and the complications of each procedure. Ninety-four percent of male infants in the TUC group and 77.3% in the SPA group were uncircumcised (P = 0.1). The mean (SD) of the PIPP pain scores did not differ between groups (9.95 ± 3.7 in SPA and 9.64 ± 3.2 in TUC, P = 0.6). The duration of TUC was longer. Both methods can be used to collect urine from neonates, but the difficulty of performing TUC on females and uncircumcised males should be considered.