Nicotinamide containing cofactors are ubiquitous in biological systems. As a result, when studying nicotinamide dependent systems in vitro or in vivo, isotopically labeled nicotinamide cofactors are often used to reveal the mechanism of the biological system of interest. The reduced form of these cofactors is labile and is sensitive to light, oxygen, pH, temperature and other factors. Consequently, their synthesis, purification, analysis, and preservation have been an ongoing challenge. Due to these stability issues, studies using nicotinamide cofactors, especially those utilizing the reduced forms, have been relegated to repetitive cycles of synthesizing the needed material just prior to experimental use. Such practice is not practical in studies utilizing complex isotopic labeling patterns. Many of the efforts that have been reported for synthesis, separation and long-term preservation of the various nicotinamide cofactors, led to procedures that are no longer practical or cost effective while others present the best solutions available today. Some of these efforts are presented and compared in this mini-review. Herein we describe our analytical and synthetic methodologies for the synthesis, separation, purification, analysis and long term preservation of labile nicotinamide cofactors, which achieve excellent versatility, yields, purity, HPLC resolution, and long term stability.