<p>Patients with myelofibrosis (MF) experience debilitating symptoms that negatively impact health-related quality of life and functioning. The Myelofibrosis Symptom Assessment Form version 4.0 (MFSAF v4.0) is a 7-item questionnaire assessing the burden of MF symptoms, including fatigue, night sweats, pruritus, abdominal discomfort, pain under the left ribs, early satiety, and bone pain. This study evaluated the content validity of the MFSAF v4.0 in Janus kinase (JAK) inhibitor–experienced participants with MF. Qualitative interviews were conducted with 20 symptomatic (MFSAF Total Symptom Score ≥ 10) participants from the US (<i>n</i> = 5) and UK, Germany, Italy, Poland, and Spain (<i>n</i> = 3 each). The interviews began with concept elicitation to allow patients to spontaneously report important symptoms. Next, content validation determined if the MFSAF v4.0 items and numeric rating scale (NRS) were relevant, important, and understandable. Overall, participants showed a clear understanding of the MFSAF v4.0, and no more than 3 (15%) mentioned that a given item was not relevant to their experience. Participants consistently felt that it was easy to select an answer on the NRS and often indicated that an improvement of 2 to 4 points was meaningful. These findings support the MFSAF v4.0 as a content-valid measure of selected disease-related symptoms for patients with JAK inhibitor–experienced MF.</p>