Commonly used antibiotics exert their effect predominantly on rapidly growing bacterial cells, yet growth dynamics taking place during infection in a complex host environment remain largely unknown. Hence, means to measure in situ bacterial growth rate is essential to predict the outcome of antibacterial treatment. We have recently validated chromosome replication as readout for in situ bacterial growth rate during Escherichia coli infection in the mouse peritonitis model. By the use of two complementary methods (qPCR and fluorescence microscopy) for differential genome origin and terminus copy number quantification, we demonstrated the ability to track bacterial growth rate, both on a population average and on a single-cell level; from one single biological specimen. Here, we asked whether the in situ growth rate could predict antibiotic treatment effect during infection in the same model. Parallel in vitro growth experiments were conducted as proof-of-concept Our data demonstrate that the activity of commonly used antibiotics ceftriaxone and gentamicin correlated with pre-treatment bacterial growth rate; both drugs performing better during rapid growth than during slow growth. Conversely, ciprofloxacin was less sensitive to bacterial growth rate, both in a homogenous in vitro bacterial population and in a more heterogeneous in vivo bacterial population. The method serves as a platform to test any antibiotic's dependency upon active in situ bacterial growth. Improved insight into this relationship in vivo could ultimately prove helpful in evaluating future antibacterial strategies.