Abstract To increase our basic understanding of the ecology and evolution of conjugative plasmids, we need a reliable estimate of their rate of transfer between bacterial cells. However, accurate estimates of plasmid transfer have remained elusive due to biological and experimental complexity. Current methods to measure transfer rate can be confounded by many factors. A notable example involves plasmid transfer between different strains or species where the rate that one type of cell donates the plasmid is not equal to the rate at which the other cell type donates. Asymmetry in these rates has the potential to bias or constrain current transfer estimates, thereby limiting our capabilities for estimating transfer in microbial communities. Inspired by the classic fluctuation analysis of Luria and Delbrück, we develop a novel approach, the Luria-Delbrück method (‘LDM’), for estimating plasmid transfer rate. Our new approach embraces the stochasticity of conjugation departing from the current deterministic population dynamic methods. In addition, the LDM overcomes obstacles of traditional methods by not being affected by different growth and transfer rates for each population within the assay. Using stochastic simulations and experiments, we show that the LDM has high accuracy and precision for estimation of transfer rates compared to the most widely used methods, which can produce estimates that differ from the LDM estimate by orders of magnitude. Significance Statement Conjugative plasmids play significant roles in the ecology and evolution of microbial communities. Notably, antibiotic resistance genes are often encoded on conjugative plasmids. Thus, conjugation—the transfer of a plasmid copy from one cell to another—is a common way for antibiotic resistance to spread between important clinical pathogens. For both public health modeling and a basic understanding of microbial population biology, accurate estimates of this fundamental rate are of great consequence. We show that widely used methods can lead to biased estimates, deviating from true values by several orders of magnitude. Therefore, we developed a new approach, inspired by the classic fluctuation analysis of Luria and Delbrück, for accurately assessing the rate of plasmid conjugation under a variety of conditions.