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Abstract Dinitrogen (N₂) fixation is an important source of biologically reactive nitrogen (N) to the global ocean. The magnitude of this flux, however, remains uncertain, in part because N₂fixation rates have been estimated following divergent protocols and because associated levels of uncertainty are seldom reported—confounding comparison and extrapolation of rate measurements. A growing number of reports of relatively low but potentially significant rates of N₂fixation in regions such as oxygen minimum zones, the mesopelagic water column of the tropical and subtropical oceans, and polar waters further highlights the need for standardized methodological protocols for measurements of N₂fixation rates and for calculations of detection limits and propagated error terms. To this end, we examine current protocols of the¹⁵N₂tracer method used for estimating diazotrophic rates, present results of experiments testing the validity of specific practices, and describe established metrics for reporting detection limits. We put forth a set of recommendations for best practices to estimate N₂fixation rates using¹⁵N₂tracer, with the goal of fostering transparency in reporting sources of uncertainty in estimates, and to render N₂fixation rate estimates intercomparable among studies.