Background and study aims Single-incision needle-knife (SINK) biopsy is a diagnostic method for acquiring tissue samples for subepithelial lesions (SELs). A single linear incision is made in the overlying mucosa and tissue samples are obtained by passing conventional biopsy forceps through the opening and deep into the lesion. The aim of this study was to describe the efficacy and safety of this technique. Patients and methods Consecutive patients who underwent SINK biopsy for an upper gastrointestinal SEL between October 2013 and September 2015 were retrospectively reviewed. Results Forty-nine patients underwent 50 SINK biopsies. Sufficient sampling for a definite pathologic diagnosis was obtained in 42 (86 %) cases, with 91 % (40/44) having sufficient sample to perform immunohistochemistry when deemed clinically relevant. Of the 26 patients with prior non-diagnostic biopsies or FNA, a specific diagnosis was obtained in 85 % (22/26). There were no significant adverse events. Conclusions SINK biopsy is a safe and feasible strategy for obtaining a definitive tissue diagnosis with immunohistochemistry for SELs.