Background: Lumbar puncture is a common procedure in many specialties. The procedure serves to diagnose life-threatening conditions, often requiring rapid performance. However, junior doctors possess uncertainties regarding performing the procedure and frequently perform below expectations. Hence, proper training and assessment of performance is crucial before entering clinical practice. Objective: To develop and collect validity evidence for an assessment tool for lumbar puncture performance, including a standard to determine when trainees are ready for clinical practice. Design: Development of a new tool, based on clinician interviews and a literature review, was followed by an explorative study to gather validity evidence. Participants and Main Measures: We interviewed 12 clinicians from different specialties. The assessment tool was used to assess 11 doctors at the advanced beginners’ level and 18 novices performing the procedure in a simulated, ward-like setting with a standardized patient. Procedural performance was assessed by three content experts. We used generalizability theory to explore reliability. The discriminative ability of the tool was explored by comparing performance scores between the two groups. The contrasting groups method was used to set a pass/fail standard and the consequences of this was explored. Key results: The interviews identified that in addition to the technical aspects of the procedure, non-technical elements involving planning and conducting the procedure are important. Cronbach’s alpha = 0.92, Generalizability-coefficient was 0.88 and a Decision-study found one rater was sufficient for low-stakes assessments (G-coefficient 0.71). The discriminative ability was confirmed by a significant difference between the mean scores of novices, 40.9 (SD 6.1) and of advanced beginners, 47.8 (SD 4.0), p = 0.004. A standard of 44.0 was established which was consistent with the raters’ global judgments of pass/fail. Conclusion: We developed and demonstrated strong validity evidence for the lumbar puncture assessment tool. The tool can be used to assess readiness for practice.