Recently, neoadjuvant treatment has turned out to be a feasible alternative for individuals suffering from locally advanced breast cancer. The neoadjuvant therapy is a type of chemotherapy that is given either before or after surgeries to diminish a tumor and minimize the likelihood of recurrence. This article demonstrates the development of a unique bioanalytical validated sensitive method by means of an ultra high performance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS) approach for the concurrent estimation of neoadjuvant treatments including 5-Fluorouracil, Doxorubicin, and Capecitabine in rat plasma. Samples were prepared using the fine minor QuEChERS process and analyzed using a Shimadzu-C18 column via an isocratic separation. Acetonitrile:water in the ratio of (30:70) (both containing 0.1 percent formic acid v/v) was the mobile phase employed at a flow rate of 0.20 mL/min. At concentrations of 50.00–500.00 ng/mL for 5-Fluorouracil, 25.00–500.00 ng/mL for Doxorubicin, and 5.00–100.00 ng/mL for Capecitabine, the procedure was shown to be linear. The limit of detection (LOD) was assessed in ng/mL and varied from 1.33 to 13.50. Relative standard deviations for precision were below 2.47 percent over the whole concentration range. For all analytes, the average recovery rate varied from 73.79 to 116.98 percent. A preliminary pharmacokinetic study was successfully performed in real rats to evaluate the procedure efficiency.