We report the first Raman spectroscopic study of propargyl-linked dihydrofolate reductase (DHFR) inhibitors being taken up by wild type Escherichia coli, Klebsiella pneumoniae, and Staphylococcus aureus cells. A novel protocol is developed where cells are exposed to the fermentation medium containing a known amount of an inhibitor. At a chosen time point, the cells are centrifuged and washed to remove the extracellular compound, then frozen and freeze-dried. Raman difference spectra of the freeze-dried cells (cells exposed to the drug minus cells alone) provide spectra of the compounds inside the cells, where peak intensities allow us to quantify the number of inhibitors within each cell. A time course for the propargyl-linked DHFR inhibitor UCP 1038 soaking into E. coli cells showed that penetration occurs very quickly and reaches a plateau after 10 min exposure to the inhibitor. After 10 min drug exposure, the populations of two inhibitors, UCP 1038 and UCP 1089, were ∼1.5 × 106 molecules in each E. coli cell, ∼4.7 × 105 molecules in each K. pneumonia cell, and ∼2.7 × 106 in each S. aureus cell. This is the first in situ comparison of inhibitor population in Gram-negative and Gram-positive bacterial cells. The positions of the Raman peaks also reveal the protonation of diaminopyrimidine ring upon binding to DHFR inside cells. The spectroscopic signature of protonation was characterized by binding an inhibitor to a single crystal of DHFR.