Techniques to evaluate renal function at the single nephron level have been instrumental and indispensible in furthering our understanding of the mammalian kidney. Techniques that were first introduce in the 1920s, and later refined in the 1950s and 1960s, permit sophisticated interrogation of glomerular filtration and hemodynamics, and tubular epithelial transport activity. Much of what we know about the physiology and pathophysiology of the kidney has been produced or, to some degree, confirmed by renal micropuncture. While micropuncture is perhaps not as widely employed as before, it remains an essential tool for comprehensive evaluation of kidney function, particularly in this age of genetically pliable experimental models. This review aims to provide a introduction to common methodologies and approaches used to conduct micropuncture experiments. Topics covered include instrumentation and equipment, pipet fabrication techniques, animal preparation, and experimental procedures for evaluating single nephron hemodynamics and tubular function. © 2012 American Physiological Society. Compr Physiol 2:621‐637, 2012.