Background The concept of segmental lumbar spine instability is controversial with varying definitions. However, suspicion of instability is used as an indication for vertebral fusion surgery in patients with lumbar degenerative spondylolisthesis and spinal stenosis. The objectives of this systematic review are to describe, the definitions of segmental instability in the degenerative lumbar spine, in studies of patients with lumbar spinal stenosis and/or lumbar degenerative spondylolisthesis, involving diagnostic imaging. And to describe which of the measurable radiological variables used to define instability have been validated against dynamic flexion-extension radiographs. Method Systematic review conducted according to the Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) guidelines. Systematic literature search will be conducted in MEDLINE and EMBASE databases, supplemented by search in Cochrane Library. International Clinical Trials Registry Platform Search Portal and ClinicalTrials.gov will be searched for ongoing or recently completed trials. PROSPERO (International Prospective Register of Systematic Reviews) will be searched before initiating this review. In addition, a gray literature search will be conducted. Studies eligible for inclusion are clinical and biomechanical studies on adult patients with degenerative spondylolisthesis and spinal stenosis and surgical treatment hereof, studies involving diagnostic imaging. We will include studies giving a definition or describing patho-anatomical findings of segmental instability or any synonym or antonym hereof. Two reviewers will independently screen articles, involving a third reviewer as referee. Screening process and data extraction conducted using Covidence software. Data synthesis presented in results checklist and systematic narrative synthesis. Data synthesis on secondary objective by contingency table. Discussion With this systematic review we want to contribute to evidence based treatment planning of lumbar degenerative spondylolisthesis with spinal stenosis. We also aim to present our result in a comprehensive manner, to encourage consensus regarding the definition of segmental instability. Registration This protocol was submitted to the International Prospective Register of Systematic Reviews (PROSPERO) on 29. April 2020, submission ID: 182827