Background: Sputum has previously attracted attention as a potential medium for detecting genetic and epigenetic alterations in early-stage lung cancer. Recent progress in advanced non-small cell lung cancer (aNSCLC) has highlighted plasma as an option in scenarios where tissue specimens are unavailable. In this study, we investigated the feasibility of mutation profiling with sputum from aNSCLC patients. Methods: Matched tissue (TIS), whole blood, and either induced or spontaneous sputum were collected from 42 prospectively enrolled, previously untreated patients with stage IIIB-IV NSCLC. Eight and two milliliters of whole blood and sputum were respectively used for preparation of plasma (PLA) and sputum supernatant (SPU). Capture-based targeted sequencing of TIS, PLA and SPU samples was performed with a panel of 520 cancer-related genes. Results: The cohort had a median age of 59 (range 26-79) with a majority of men (31/42). Thirty-five patients were diagnosed with lung adenocarcinoma (LUAD) and the remaining seven with squamous cell carcinoma (LUSC). There were more non-smokers (26/42) than smokers. Most patients (34/42) had metastatic disease. First, we assessed the performance of three sample types in terms of key quality control indices. PLA and SPU showed maximum allelic fraction that were both significantly smaller than that of TIS. Subsequently compared was positive detection rate, TIS, PLA, and SPU achieved respective rates of 92.9%, 71.4%, and 59.5%, revealing comparable levels between PLA and SPU samples (p = 0.36). Rates of detection were highly similar between induced (54.6%) and spontaneous sputum (61.3%), but were higher in LUSC (85.7%) than in LUAD patients (54.3%) although this difference was not statistically significant (p = 0.21). Furthermore, detection rates rose in smokers (81.3% for PLA and SPU, 100% for TIS). Next, using TIS as the gold standard, we compared the sensitivity of detecting eight established driver alterations in NSCLC with the two liquid biopsies. SPU and TIS achieved respective levels of 50.0% and 70.0%, which, similar to detection rate, improved to 88.9% (8/9) and 77.8% (7/9) in the smokers subgroup. Importantly, detection of altered KRAS and ALK was more sensitive with SPU (6/6, 2/3, entire cohort) than with PLA (5/6, 1/3). For EGFR mutation, which occurred mainly in nonsmokers, the sensitivity was considerably lower in SPU (4/14, entire cohort) than in PLA (12/14). Detection of mutant MET, however, appeared difficult for both biopsies (0/4). In addition, there was a high level of correlation between estimates of tumor mutational burden based on PLA and on SPU (Pearson r² = 0.58). Conclusions: Our study demonstrated that for aNSCLC patients, sputum supernatant demonstrates comparable performance as plasma. Sputum is therefore a viable alternative for profiling for established driver alterations in aNSCLC, especially in smokers.