ALK inhibitors have improved the therapeutic management of patients with ALK-rearranged advanced non–small cell lung cancers (NSCLC). Several diagnostic methods, mainly fluorescent in situ hybridization (FISH) and immunohistochemistry (IHC), can be used as single or combined tests to detect the so-called “ALK-positive” NSCLC. Intersample and intermethod discrepancies could cause issues with therapeutic consequences in ALK testing. In this article, we report a case series of our real-life experience and issues in combining FISH and IHC in multiple tumor samples per patient to diagnose and treat a subset of “ALK-positive” patients with NSCLC. Among analyses conducted in 40 samples of 18 patients with advanced lung adenocarcinomas, we retrospectively encountered 10 patients with intersample-concordant ALK FISH and IHC results. Discrepant results about FISH and/or IHC were noted between different samples in 8 patients. Therapeutic responses were observed in 5 of 10 crizotinib-treated patients including 1 patient with ALK FISH+ IHC− status and 1 patient with ALK FISH− IHC+ status. Our data highlight the difficulty to predict the response/nonresponse to crizotinib therapy, in patients with advanced NSCLC, not only on the basis of single and multiple tumor samples, but also on the basis of single and combined diagnostic methods.