Smartphone accelerometry has potential to provide clinicians with specialized gait analysis not available in most clinical settings. The Gait&Balance Application (G&B App) uses smartphone accelerometry to assess spatiotemporal gait parameters under two conditions:​ walking looking straight ahead and walking with horizontal head turns. This study investigated the validity of G&B App gait parameters compared with the GAITRite® pressure-sensitive walkway. Healthy young and older adults (age range 21–85 years) attended a single session where a smartphone was secured over the lumbosacral junction. Data were collected concurrently with the app and GAITRite® systems as participants completed the two walking conditions. Spatiotemporal gait parameters for 54 participants were determined from both systems and agreement evaluated with partial Pearson’s correlation coefficients and limits of agreement. The results demonstrated moderate to excellent validity for G&B App measures of step time (r_p 0.97, 95 % CI [0.96, 0.98]), walking speed (r_p 0.83 [0.78, 0.87]), and step length (r_p 0.74, [0.66, 0.80]) when walking looking straight ahead, and results were comparable with head turns. The validity of walking speed and step length measures was influenced by sex and height. G&B App measures of step length variability, step time variability, step length asymmetry, and step time asymmetry had poor validity. The G&B App has potential to provide valid measures of unilateral and bilateral step time, unilateral and bilateral step length, and walking speed, under two walking conditions in healthy young and older adults. Further research should validate this tool in clinical conditions and optimise the algorithm for demographic characteristics.