Background YouTube has become an important educational platform. For biomedical students the site is a primary information source for informational and instructional videos on a variety of medical topics.This study examined the educational quality of instructional lumbar puncture procedure videos on YouTube.Methods The search strings “lumbar puncture medical procedure” and “spinal tap medical procedure” were used. The first 100 videos for each search string were assessed for eligibility. Video characteristics were documented. To evaluate the popularity of the videos the Video Power Index was calculated. Four validated checklists were used for video assessment: Lumbar Puncture Assessment Tool, reliability score, Global Quality Score, Attractiveness and Understandability score.Results Lumbar puncture videos had a median ratio of 8,90 views/day, 0,04 likes/day and a video power index of 14,44, which was lower compared to other categories of instructional videos (CPR and cake baking). The mean LumPAT score was 34/55, with only three videos scoring at least 44/55, the pass ratio of this assessment. The median reliability score was 3/4, the median GQS 3/5 (moderate quality) and the median AU score 6,5/8. Significant correlations were found between the LumPAT score and the video length (p = 0,003), the GQS (p < 0,001) and the AU score (p = 0,01).Conclusions In this study, only three out of the 23 videos passed the LumPAT score. The majority of the instructional videos are not suitable for the training of health care professionals and students in performing a lumbar puncture procedure.