Chloroplasts, originating from ancestral cyanobacteria, are essential plastids involved in photosynthesis and other key metabolic processes, making them vital components of plant cells. Chloroplast transformation is a preferred alternative to nuclear transformation in plant genetic engineering due to its numerous advantages and positive outcomes. This chapter explores the available various chloroplast transformation vectors, cloning the gene of interest (GOI) in the chloroplast transformation vector with step-by-step protocol, and preparation of the vector for chloroplast transformation of tobacco using leaf disc explants through a biolistic method for efficient gene delivery. Additionally, it highlights how crucial some factors are for the effective integration and expression of the chloroplast genome by DNA-coated particle bombardment, including microcarrier type, DNA concentration, helium accelerating pressure, leaf layout and firing distance, and duration of pretreatment for explants. Further explored the generation of homoplasmic plants through re-regeneration with antibiotic selection confirmed correct cassette insertion and homoplasmy using PCR and Southern blot analyses and verified maternal inheritance of chloroplast-transferred genes by examining green and albino seedlings from reciprocal crosses between control and transplastomic tobacco plants.