Antibiotic resistance is a major global health concern. The increased use of herbicides may lead to multiple antibiotic resistance in bacteria. Conventional techniques for diagnosing antibiotic resistance are laborious, time-intensive, expensive, and lack information about antibiotic susceptibility. On the other hand, Raman spectroscopy is a rapid, label-free, noninvasive alternative to traditional techniques to detect antibiotic resistance. In this study, two popular herbicides 2,4-dichlorophenoxy acetic acid (2,4-D) and N-(phosphonomethyl)glycine (glyphosate) were used to study their effects on the emergence of antibiotic resistance. The Escherichia coli wild-type (WT) MG1655 strain and two isogenic mutants, Δlon and ΔacrB, were used together with Raman spectroscopy. The WT E. coli is sensitive to antibiotics, but exposure to both herbicides induces antibiotic resistance. Using an excitation wavelength of 785 nm, the intensity ratios (e.g., I740/I785, I740/I1003, I1480/I1445, I2934/I2868, and I2934/I2845) were identified as biomarkers to study the induction of antibiotic resistance in bacteria but not NaCl-mediated stress. Using an excitation wavelength of 633 nm, the peak intensity at 740 cm-1 assigned to cytochrome bd decreases under antibiotic stress but increases upon exposure to both herbicides and antibiotics, indicating the development of resistance. Thus, this study can be applied to monitor antibiotic resistance using Raman spectroscopy.