Background There are no recommendations on the optimal dosing for cryoablation of atrial fibrillation (Cryo-AF). Objective The purpose of this study was to develop and prospectively test a Cryo-AF dosing protocol guided exclusively by time-to-pulmonary vein (PV) isolation (TT-PVI) in patients undergoing a first-time Cryo-AF. Methods In this multicenter study, we examined the acute/long-term safety/efficacy of Cryo-AF using the proposed dosing algorithm (Cryo-AFDosing; n=355) against a conventional, nonstandardized approach (Cryo-AFConventional; n=400) in a nonrandomized fashion. Results Acute PV isolation was achieved in 98.9% of patients in Cryo-AFDosing (TT-PVI = 48 ± 16 seconds) vs 97.2% in Cryo-AFConventional (P = .18). Cryo-AFDosing was associated with shorter (149 ± 34 seconds vs 226 ± 46 seconds; P Conclusion A novel Cryo-AF dosing algorithm guided by TT-PVI can help individualize the ablation strategy and yield improved procedural endpoints and efficiency as compared to a conventional, nonstandardized approach.