The rate of mutation refers to the probability that a unit length of DNA (generally a base pair) mutates with time. Fluctuation analysis or mutant accumulation assays applied to phenotypic changes measure mutation rates of cells. However, only a few phenotypic changes indicative of mutations are known thus limiting the analysis to those rare genes. Direct sequencing overcomes the limitations imposed by phenotypic analysis but is limited by the extensive number of clones or cells that have to be analyzed in fluctuation or mutant accumulation assays. We propose a strategy to determine the rate of mutation of a gene by limited direct sequencing of a few single cells of a defined lineage. To accomplish this, we determined the average number of mutations per position in each DNA length sequenced from the proportion of the non-mutated positions, according to the Poisson process and/or the Taylor series. Measuring the rate of mutation by direct sequencing of genes does not require ascertaining a phenotype and can be applied to any area of the genome in a cell. The approach avoids fluctuation errors.