Background: Step counts are increasingly used in public health and clinical research to assess wellbeing, lifestyle, and health status. However, estimating step counts using commercial activity trackers has several limitations, including a lack of reproducibility, generalizability, and scalability. Smartphones are a potentially promising alternative, but their step-counting algorithms require robust validation that accounts for temporal sensor body location, individual gait characteristics, and heterogeneous health states. Objective: Our goal was to evaluate an open-source step-counting method for smartphones under various measurement conditions against step counts estimated from data collected simultaneously from different body locations (internal validation), manually ascertained ground truth (manual validation), and step counts from a commercial activity tracker (Fitbit Charge 2) in patients with advanced cancer (wearable validation). Methods: We used eight independent datasets collected in controlled, semi-controlled, and free-living environments with different devices (primarily Android smartphones and wearable accelerometers) carried at typical body locations. Five datasets (N=103) were used for internal validation, two datasets (N=107) for manual validation, and one dataset (N=45) used for wearable validation. In each scenario, step counts were estimated using a previously published step-counting method for smartphones that uses raw sub-second level accelerometer data. We calculated mean bias and limits of agreement (LoA) between step count estimates and validation criteria using Bland-Altman analysis. Results: In the internal validation datasets, participants performed 751.7+/-581.2 (mean+/-SD) steps, and the mean bias was -7.2 steps (LoA -47.6, 33.3) or -0.5%. In the manual validation datasets, the ground truth step count was 367.4+/-359.4 steps while the mean bias was -0.4 steps (LoA -75.2, 74.3) or 0.1 %. In the wearable validation dataset, Fitbit devices indicated mean step counts of 1931.2+/-2338.4, while the calculated bias was equal to -67.1 steps (LoA -603.8, 469.7) or a difference of 0.3 %. Conclusions: This study demonstrates that our open-source step counting method for smartphone data provides reliable step counts across sensor locations, measurement scenarios, and populations, including healthy adults and patients with cancer.