BACKGROUND: Chronic ankle instability (CAI) is associated with defective posture control and balance; thus, a proper assessment of these impairments is necessary for effective clinical decision-making. There is a need for portable, valid, and reliable methods to facilitate the easy collection of real-world data, such as mobile phones. RESEARCH QUESTION: Is the smartphone "MyAnkle" application valid and reliable in assessing balance in patients with CAI and healthy volunteers? METHODS: This was a cross-sectional study. Sixty-five participants completed two assessment sessions, including 31 patients (n = 41 ankles with CAI and 21 asymptomatic ankles) and 34 healthy volunteers (n = 68 ankles). In each session, dynamic single-leg stance balance was measured simultaneously using the "MyAnkle" application and the Biodex balance system (BBS) version 3. Testing was conducted at three levels of BBS difficulty-4 (D4, hard, loose platform), 6 (D6, moderate), and 8 (D8, easy, stiffer platform)-and repeated with opened and closed eyes. Both limbs were tested in a random order by two independent blinded assessors. RESULTS: The two devices showed significant poor-to-moderate correlations when eyes were closed (p < 0.05). For discriminant validity, the application did not distinguish the two study groups in all tested conditions (p > 0.05), whereas the BBS weakly to moderately distinguished the dominant limbs in the two groups at all difficulty levels with eyes-open and at D8 with eyes-closed regardless to limb dominance. For reliability, a significantly poor to moderate inter-session reliability was noted for the two devices. SIGNIFICANCE: "MyAnkle" application is valid in assessing balance in patients with CAI when the eyes are closed. However, similarly to BBS, its one-week test-retest reliability may be insufficient for accurate follow-up of balance changes and need to be interpreted with caution. Future studies need to establish its inter-tester reliability and its usefulness in telerehabilitation.