<p>Chloroplasts are essential organelles in plant cells that carry out photosynthesis and support numerous metabolic functions. They also respond dynamically to environmental stress, playing a central role in plant adaptation. Understanding chloroplast behavior under such conditions often requires the isolation of intact chloroplasts, which allows direct examination of their structure and function. While Percoll density gradient centrifugation is commonly used for chloroplast isolation, existing protocols may not perform consistently across plant species, including those rich in secondary metabolites, which limits the isolation of intact and pure chloroplasts. In this study, we present an improved method for isolating intact chloroplasts from a range of plant species. The protocol includes optimized centrifugation speeds and adjusted loading volumes over the Percoll gradient to enhance the recovery and purity of intact chloroplasts. Isolated chloroplasts were evaluated using fluorescence microscopy, confocal imaging, and subcellular fractionation assays to confirm their structural integrity. Our results show that this method reliably yields intact and pure chloroplasts from multiple species, including <i>Nicotiana benthamiana</i>, <i>Arabidopsis thaliana</i>, <i>Oryza sativa</i>, <i>Solanum lycopersicum</i>, and secondary metabolite-rich plants <i>Stevia rebaudiana</i>, <i>Camellia sinensis</i>, and <i>Sambucus javanica,</i> indicating its broader applicability.</p>