Tyrosine kinase inhibitor (TKI) discontinuation in chronic myeloid leukemia (CML) has become part of routine care for patients with a sustained deep molecular response (DMR). Approximately 50% experience a molecular relapse upon TKI cessation. Most of them quickly regain DMR upon TKI resumption. Whether these patients can achieve a second treatment-free remission (TFR) remains unclear. DAstop2 (ClinicalTrials.gov ID: NCT03573596) is a prospective study including patients with a failed first TFR attempt re-treated with any TKI for ≥ one year. Upon entering the study, patients received the TKI dasatinib for additional two years. Patients with sustained DMR for ≥1 year qualified for a second TKI stop. Ninety-four patients were included between Oct 2017-Dec 2021. At the time of data analysis, 62 patients had attempted a 2 stop. After a median follow-up of 27 months from 2 stop, TFR rates were 61, 56 and 46% at 6, 12 and 24 months respectively. No progression to advanced stage disease was seen and 87% had re-achieved MR within a median of 3 months from TKI re-initiation. In summary, we show that a 2 TFR attempt after dasatinib treatment is safe, feasible and TFR rates seem in the range of those reported in trials of a first TKI stop.