Since lipid nanoparticles (LNP) have emerged as a potent drug delivery system, the objective of this study was to develop and optimize a robust high-performance liquid chromatography with charged aerosol detectors (HPLCCAD) method to simultaneously quantify different lipids in LNPs using the analytical quality by design (AQbD) approach. After defining analytical target profile (ATP), critical method attributes (CMAs) were established as a resolution between the closely eluting lipid peaks and the total analysis time. Thus, potential high-risk method parameters were identified through the initial risk assessment. These parameters were screened using Plackett–Burman design, and three critical method parameters (CMPs)—MeOH ratio, flow rate, and column temperature—were selected for further optimization. Box–Behnken design was employed to develop the quadratic models that explain the relationship between the CMPs and CMAs and to determine the optimal operating conditions. Moreover, to ensure the robustness of the developed method, a method operable design region (MODR) was established using the Monte Carlo simulation. The MODR was identified within the probability map, where the risk of failure to achieve the desired CMAs was less than 1%. The optimized method was validated according to the ICH guidelines (linearity: R2 > 0.995, accuracy: 97.15–100.48% recovery, precision: RSD < 5%) and successfully applied for the analysis of the lipid in the LNP samples. The development of the analytical method to quantify the lipids is essential for the formulation development and quality control of LNP-based drugs since the potency of LNPs is significantly dependent on the compositions and contents of the lipids in the formation.