Pioglitazone, a PPAR-gamma activator used to diagnose hyperglycemia, was studied for its stereoselective deposition and active enantiomers in female albino Wistar rats. In accordance with USFDA recommendations, a bioanalytical technique was employed to assess the segregation of pioglitazone enantiomers in rat plasma with glimepiride as an internal standard. A Phenomenox i-Amylose-3 column (150 mm × 4.6 mm) of 5 µm was used for high-performance liquid chromatography (HPLC) with a mobile phase of 10 mM ammonium acetate buffer in Millipore water and acetonitrile in 60:40 (v/v) admixture with column temperature 35 °C, wavelength 265 nm, and flow rate 0.6 mL/min, respectively. Pioglitazone-S, Pioglitazone-R, and the internal standard had retention times of 3.1, 7.4, and 1.7 min, respectively. The study found that within-run and between-run precision ranged from 0.1606–0.9889% for Pioglitazone-R and from 0.2080–0.7919% for Pioglitazone-S, while the accuracy ranged from 99.86 to 100.36% for Pioglitazone-R and 99.84 to 99.94% for Pioglitazone-S. In addition, a non-radioactive glucose uptake assay was employed to examine the enantiomers in 3T3-L1 cell lines by flow cytometry. Significant differences were demonstrated in Cmax, AUClast (h*μg/mL), AUCINF obs (h*μg/mL), and AUC%Extrap obs (%) of Pioglitazone-R and S in female albino Wistar rats, suggesting enantioselectivity of pioglitazone.