Measuring bacterial growth rates is routine, however, determining growth rates during infection in host has been more challenging. Peak-to-trough ratio (PTR) is a technique for studying microbial growth dynamics, calculated using the ratio of replication origin () copies to that of the terminus (), as originally defined by whole genome sequencing (WGS). WGS presents significant challenges in terms of expense and data analysis complexity due to the presence of host DNA in the samples. Here, we used multiplexed PCR with fluorescent probes to estimate bacterial growth rates based on the abundance of - and -adjacent loci, without the need for WGS. We establish the utility of this approach by comparing growth rates of the uropathogenic (UPEC) strain HM86 by WGS (PTR) and qPCR to measure the equivalent (O:T ). We found that PTR and O:T were highly correlated and that O:T reliably predicted growth rates calculated by conventional methods. O:T was then used to calculate the growth rates in urine, bladder, and kidneys collected over the course of a week from a murine model of urinary tract infection (UTI). These analyses revealed that growth rate of UPEC strains gradually increased during the early stages of infection (0-6h), followed by a slow decrease in growth rates during later time points (1-7 days). This rapid and convenient method provides valuable insights into bacterial growth dynamics during infection and can be applied to other bacterial species in both animal models and clinical infections.