<p>A sensitive analytical method was developed and validated for the simultaneous quantification of insulin glargine and its active metabolites M1 and M2 in human K₂EDTA plasma. Plasma sample extraction was performed using an optimized mixed-mode solid-phase extraction (SPE) procedure, followed by analysis using liquid chromatography–tandem mass spectrometry (LC–MS/MS API 6500+) in positive electrospray ionization mode. The method specificity, linearity, sensitivity, recovery, accuracy, precision, IS-normalized matrix factor, matrix effect, and stability, were systematically validated as per ICH M10 guidelines. The method exhibited high specificity, with no significant interference at the retention times of analytes and internal standards. The calibration curves showed linearity over the concentration range of approximately 70–2200 pg/mL, with regression coefficients (r²) ≥ 0.995. The lower limit of quantification was approximately 70 pg/mL for analyte and metabolites. Observed intra-day and inter-day precision (2.93–11.27%) and accuracy were well within acceptable regulatory thresholds. The overall mean analyte recovery was consistent (61–63%) with low variability (CV &lt; 5%) across three quality control levels. Internal standard normalization method demonstrated minimal matrix effect with a %CV ≤ 7.74%. The validated method thus delivers high sensitivity and reproducible quantification while maintaining a notably simplified sample preparation workflow without the need for immunoaffinity purification (IAP). The designed and tested method is valid and applicable for pharmacokinetic, bioavailability, and bioequivalence studies of insulin glargine and its metabolites.</p>