BackgroundEndotracheal aspiration is a painful nociceptive procedure. There is still a gap in the literature on studies to determine the pain level and nursing interventions for aspiration. ObjectivesThis study evaluated pain during endotracheal aspiration and examined the factors affecting pain. MethodsThis prospective observational study was conducted with 105 inpatients meeting the inclusion criteria in the internal intensive care unit of a public hospital. Two hundred ten aspiration procedures were monitored for pain and other variables. ASPMN 2019 Position Statement recommendations were followed in designing the study and determining the procedure. The pain score range obtained from The Critical Care Pain Observation Tool was 0–8. A score of 2 or more is considered to indicate the presence of pain. The primary outcome measures were pain associated with endotracheal aspiration and affecting factors in this study. The generalised linear mixed model established for aspiration procedure-associated pain and affecting factors was analysed. ResultsPatients' mean pain score was 1.24 ± 2.05 before, 3.07 ± 2.17 during, and 2.35 ± 1.94 after aspiration and 0.89 ± 1.40 at 15 min after aspiration. The pain rate was 26.1% before, 71% during, and 60.9% after the aspiration procedure and 18.8% after 15 min. There was a statistically significant difference between all pain scores evaluated. The difference in aspiration-related pain scores by age, respiratory diseases, sedation status, aspiration pressure, and tube diameter was statistically significant. ConclusionsThe pain score due to aspiration procedure increased significantly in intensive care unit inpatients and is an important risk factor for patient safety. More focus is needed on the causes and measures of aspiration-related pain.