DNA damage repair proteins form foci in response to DNA damaging agents. The efficiency and integrity of the DNA repair pathway of a particular eukaryotic (mutant) strain is usually determined by the number of foci formed compared with their wild-type counterpart. Conventionally, focus number is determined visually, and this low accuracy may obscure the identification of a weaker phenotype, particularly when the output is low. Here, using the homologous recombination protein Rhp54 as an example, we present a protocol that can increase the consistency of foci identification among samples and can significantly improve the efficiency of foci quantification for large sample sizes. A similar method can be applied to other foci-forming proteins.