Lumbar puncture (LP) is a commonly performed low-risk procedure terrestrially, used diagnostically for evaluation of cerebrospinal fluid (CSF) pressure as well as for collection of CSF for analysis. NASA is investigating noninvasive means for measurement of intracranial pressure (ICP) to assess the potential contribution of elevated intracranial pressures to recently reported changes in astronauts' visual acuity and eye anatomy, known collectively as the Visual Impairment/Intracranial Pressure risk. However, many of these noninvasive technologies are still under development, have limited clinical validation, are several years away from being ready for in-flight use, or only provide qualitative rather than quantitative ICP values. Therefore, performance of in-flight LPs, as part of crewmember evaluation, has also been considered by NASA. This manuscript summarizes the unique operational considerations, constraints, concerns, and limitations of using traditional LP as an adjunct or as an alternative to noninvasive ICP measurements during spaceflight.