In the present study, L-proline derivatives and linker chains are designed, synthesized and were characterized using spectral analysis. In the present study, 14 L-proline derivatives were synthesised among which five derivatives were consisting of symmetrical linker chains and nine derivatives are of asymmetrical linker chains. The synthesised compounds are then interacted through H-bond interactions with Topoisomerase-I (Human DNA) enzyme active sites. The docking analysis of L-proline derivative reveals that, among 14 compounds synthesised, compound IVL6 and IIIL4 were found to be more potent towards Topoisomerase-I enzyme with London dG scoring values of -12.8472 and -11.5501 respectively. All the synthesized compounds were then characterized using spectral analysis. Hence the present study forms the basis for the synthesis and characterization of L-proline derivatives as possible bis-intercalators to potentially act as anticancer agents. Key words: Topoisomerase I enzyme, L-proline derivatives, docking analysis, H-bond interactions and anticancer agents.