N-Alkyl/benzyl substituted isatin derivatives are intermediates and synthetic precursors for the preparation of biolog- ical active heterocycles. N-alkyl/benzyl isatins have showed various biological activities, such as cytotoxicity, antiviral, caspase inhibition, cannabinoid receptor 2 agonists for the treatment of neuropathic pain, etc. In this study, N-alkyl/benzyl isatin deriva- tives were synthesized from isatin and alkyl/benzyl halides in presence of K2CO3 in DMF and excellent to quantitative yields (~95%) were obtained. Isatins and benzyl-isatins were condensed with fluorescein hydrazide to form fluorescein hydrazone. All the compounds were subjected to their fragmentation behavior study using LC/MS n . N-Alkyl substituted isatin derivatives frag- mented at nitrogen-carbon (N-C) bond, hence gave daughter ion as (RN+H) + . Whereas, N-benzyl substituted isatin derivatives fragmented at carbon-carbon (C-C) bond of alkyl chain which linked with nitrogen molecules, therefore gave N-methyl frag- ments (RNCH2) + . This study demonstrated that, isatin moiety present in a small/large molecule or in a matrix of reaction mixture with/without N-alkyl/benzyl substituents can be identified by mass spectroscopic fragmentation behavior study.