The one-step nucleic acid amplification (OSNA) method allows for the quantitative evaluation of the tumor burden in resected lymph nodes (LNs) in patients with lung cancer. This technique enables to detect macro and micrometastases, facilitating the correct classification of patients for appropriate follow-up of the disease after surgery. Of 160 patients with resectable lung cancer whose LNs were examined by OSNA, H&E and CK19 IHC between July 2015 and December 2018, 110 patients with clinical stages from IA1 to IIIB were selected for follow-up. LN staging in lung cancer by pathological study led to understaging in 13.64% of the cases studied. OSNA allowed to quantify the tumor burden and establish a prognostic value. Patients with a total tumor load of ≥1650 cCP/uL were associated with a significantly increased likelihood of recurrence. Moreover, the survival of patients with <4405 cCP/uL was significantly higher than patients with ≥4405 cCP/uL. The OSNA assay is a rapid and accurate technique for quantifying the tumor burden in the LNs of lung cancer patients and OSNA quantitative data could allow to establish prognostic values for recurrence-free survival and overall survival in this type of malignancy.