AbstractBiomass estimation is central in most microbial plankton ecology studies. Numerous advances have been made that rendered this task more and more effective, from the introduction of allometric volume‐to‐carbon conversion factors to the development of submersible flow‐through imaging devices. Although several ingenious methods have been suggested for this purpose, the problem of biovolume extrapolation from two‐dimensional pictures remains. This study offers a historical and critical review of the methods for the estimation of the biovolume of microorganisms that have been proposed throughout the last four decades. It covers three main approaches—respectively based on a single equation, a set of simple or composite geometric shapes or a computational algorithm—as well as several methods featuring exclusive characteristics. Both strengths and shortcomings of each technique are analyzed, with particular emphasis on compatibility with the most recent flow‐through devices coupled with in situ imaging systems. A composite picture emerges with no such unique method, at present, suitable for every situation. This review can thus be a valuable guide for the microbial plankton ecologist who seeks the most suitable technique for a specific circumstance.