The present study reported leciplex (LPX) preparation using a simple mixing technique for 10 mL to 200 mL volumes. A D-optimal design was employed to study the effect of different variables on the characteristics of LPX and compare their impact where the batch volume (X1), mixing speed (X2), and mixing time (X3) were set as the independent variables, whereas their impact was studied for, particle size (PS; Y1), polydispersity index (PDI; Y2), zeta potential (ZP; Y3). Furthermore, the LPX batch prepared via optimized parameters was characterized for morphology by transmission electronic microscopy (TEM) and stability study. The PS (Y1) of different LPX batches were significantly affected by mixing speed (X2), while the PDI (Y2) and ZP (Y3) were influenced considerably by both mixing speed (X2) and mixing time (X3). The batch volume (X1) did not affect the characteristics of the prepared batches. The morphology of LPX was spherical and well dispersed. Furthermore, the optimized LPX batch was stable for 90 days of storage. Finally, it was shown that this fabrication method was appropriate for preparing LPX with good reproducibility and stability.