Bacterial Ghosts (BGs) received an increasing interest in the recent years for their promising medicinal and pharmaceutical applications. In this study, for the first time we introduce a new protocol for BGs production. E. coli BL21 (DE3) pLysS (Promega) was used as a model to establish a general protocol for BGs preparation. The protocol is based on using active chemical compounds in concentrations less than the Minimum Inhibition Concentration (MIC). Those chemical compounds are SDS, NaOH, and H2O2. Plackett-Burman experimental design was used to map the best conditions for BGs production. Normal and electronic microscopes were used to evaluate the BGs quality (BGQ). Spectrophotometer was used to evaluate the amount of the released protein and DNA. Agarose gel electrophoresis was used to determine the existence of any residue of DNA after each BGs preparation. Viable cells, which existed after running this protocol, were subjected to lysis by inducing the lysozyme gene carried on pLysS plasmid. This protocol is able to produce BGs that can be used in different biotechnological applications.