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Urinalysis (UA) has routinely been used as a screening tool prior to urine culture set up. BacterioScan 216Dx is an FDA-cleared semiautomated system to detect bacterial growth in urine. The aim of this study was to evaluate 216Dx in comparison to UA for diagnosis of urinary tract infection (UTI) in children. Clean-catch, unpreserved urine samples from children aged <18 years were tested by 216Dx, and positive urine samples in media were processed for direct bacterial identification by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry. Sensitivity and specificity of 216Dx and urinalysis (UA) were determined against urine culture. Of 287 urine samples obtained from children (median age, 108 months), 44.0% and 56.0% were UA positive and negative, respectively, while 216Dx detected 27% and 73% as positive and negative, respectively. Compared to culture, the overall sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of 216Dx versus UA were 92.1% versus 97.3%, 82.7% versus 63.8%, 44.8% versus 29.1%, and 98.6% versus 99.3%, respectively. Among 216Dx true-positive (TP) samples ( n = 35), 77.0% were successfully identified directly from broth by MALDI-TOF. Among urine samples that were identified as contaminated by culture ( n = 127; 44%), the 216Dx detected 93 (73.0%) as negative while UA detected 69 (54.0%) as negative. Although the sensitivities of 216Dx and UA are comparable, the specificity of 216Dx was higher than that of UA. The 216Dx can be used as an alternative/adjunct screening tool to UA to rule out urinary tract infection (UTI) in children. Compared to culture, the faster turnaround time (3 hours) of 216Dx has the potential to reduce unnecessary antibiotic use and improve patient management., (Copyright © 2019 American Society for Microbiology.)