Introduction: Alectinib is an effective second-generation ALK tyrosine kinase inhibitor (TKI) used in the first-line treatment of patients with advanced ALK-positive NSCLC. Recent studies demonstrated that the percentage of ALK-positive tumor cells in patient groups receiving crizotinib might affect outcomes. This study aimed to investigate whether the percentage of ALK-positive cells had a predictive effect in patients with advanced NSCLC who received first-line Alectinib as ALK-TKI.Materials and Methods: This retrospective study included patients with advanced-stage NSCLC who received alectinib as a first-line ALK-TKI and whose percentage of ALK-positive cells was determined by FISH at 27 different centers. Patients who received any ALK-TKI before alectinib were not included in the study. Patients were separated into two groups according to the median (40%) value of the percentage of ALK-positive cells (high-positive group ≥40% and low-positive group Results: 211 patients were enrolled (48.3% female, 51.7% male) to study. 37% (n=78) of the patients had received chemotherapy previously. After a median of 19.4 months of follow-up, the median PFS was not reached in the high-positive group (n=113) but it was 10.8 months in the low-positive group (n=98) (HR: 0.39; 95% CI, 0.25-0.60, pConclusion: Our study demonstrated that the efficacy of alectinib varies significantly across patient subgroups with different percentages of ALK-positive cells. If these findings are prospectively validated, the percentage of ALK-positive cells may be used as a stratification factor in randomized trials comparing different ALK-TKIs.