Gluconeogenesis is a complex metabolic process that involves multiple enzymatic steps regulated by myriad factors, including substrate concentrations, the redox state, activation and inhibition of specific enzyme steps, and hormonal modulation. At present, the most widely accepted technique to determine gluconeogenesis is by measuring the incorporation of deuterium from the body water pool into newly formed glucose. However, several techniques using radioactive and stable-labeled isotopes have been used to quantitate the contribution and regulation of gluconeogenesis in humans. Each method has its advantages, methodological assumptions, and set of propagated errors. In this review, we examine the strengths and weaknesses of the most commonly used stable isotopes methods to measure gluconeogenesis in vivo. We discuss the advantages and limitations of each method and summarize the applicability of these measurements in understanding normal and pathophysiological conditions.