Fluorescence in situ hybridization (FISH) is currently used to detect non-small cell lung cancer (NSCLC) patients with anaplastic lymphoma kinase (ALK) gene rearrangement, who are candidates for ALK inhibitor therapy. However, FISH may not be a practical method for screening for ALK-positive patients in a large population due to its cost and difficulty in interpretation. We investigated the role of immunohistochemistry (IHC) to screen for ALK rearrangement in advanced NSCLC. We identified 1,166 stage IIIB or IV NSCLC patients with non-squamous histology from the Seoul National University Hospital NSCLC database. To enrich ALK-positive cases, we selected 262 patients who were either EGFR wild-type or non-responders to previous EGFR tyrosine kinase inhibitors (TKI). ALK IHC and ALK FISH were performed on formalin-fixed, paraffin-embedded tissue. ALK protein was expressed in 28 (10.7%) tumors in 262 patients. ALK FISH was positive in 25 (9.5%) cases. All patients with IHC score of 3 (n=9) were FISH-positive and all patients with score of 0 (n=234) were FISH-negative. Among patients with IHC scores of 1 and 2, five (83.3%, 5/6) and eleven (84.6%, 11/13) were FISH-positive, respectively. The sensitivity and specificity of ALK IHC with intensity score of 1 or more were 100% and 98.7%, respectively. IHC can be a useful test for screening ALK FISH-positive cases in advanced NSCLC. FISH testing should be considered for advanced NSCLC patients with tumors showing mild to moderate staining for ALK by IHC to confirm ALK translocation.