Experimental evolution is an important research framework for evolutionary biologists as it allows direct testing of fundamental theories about adaptation and diversity, which often requires the tracking of genotypes or alleles over time. This however requires tools such as genetic markers, distinguishable morphological characters or genotyping, which can be time and labour intensive, and especially if high-throughput processing is necessary. Here, we present a novel approach of combining multiplex ddPCR with experimental evolution for tracking genotype frequencies in different experimental populations over time. We found this method to be both precise and accurate for detecting and quantifying targets of interest, especially as compared to traditional PCR methods. The use of multiplex ddPCR to follow frequencies can be used for a wide range of experimental evolution applications.