Plumule of Nelumbo nucifera Gaertn., a traditional Thai medicine, is primarily used for nervous disorder, insomnia, high fevers with restlessness, and cardiovascular disease. Neferine is one of the main bisbenzylisoquinoline alkaloid components in N. nucifera plumule and has been reported to possess several pharmacological properties. Standardization of plant materials is necessary for an assurance of quality, efficacy and safety based on various parameters. This study aimed to investigate the pharmacognostic specification of N. nucifera plumule and determine the neferine content using TLC densitometry and TLC image analysis. Transverse section of this crude drug showed the anatomical characteristics of epidermis, air chamber, parenchyma and vascular bundle. Additionally, the histological study of powder showed anomocytic stomata, parenchyma with starch granule and layer of cotyledons. TLC fingerprint and physicochemical constants were also established. The average values of total ash, acid insoluble ash, loss on drying, moisture content, ethanol and water extractive contents were found to be 3.86, 0.54, 9.91, 10.02, 16.54 and 28.27 g% respectively. Moreover, TLC densitometry and TLC image analysis were validated for determination of neferine content in N. nucifera plumule. The calibration range was between 0.25-3.0 µg/spot and exhibited suitable accuracy, precision and robustness in both methods. Neferine content in N. nucifera plumule were 0.496 ± 0.186 g% by TLC densitometry and 0.495 ± 0.190 g% by TLC image analysis. Additionally, N. nucifera plumule ethanolic extract, dichloromethane fractionate and ethanolic fractionate were tested for in vitro cytotoxic activity against 5 human cancer cell lines including breast ductal carcinoma (BT-474), undifferentiated lung carcinoma (CHAGO -K1), colon adenocarcinoma (SW-620), gastric carcinoma (KATO-3), hepatocarcinoma (HEP-G2) and one human normal cell line (lung fibroblast, Wi-38). N. nucifera plumule ethanolic extract, dichloromethane fractionate and ethanolic fractionate exhibited no significant activity against the five cancer cell line as well as one normal cell line with an IC50 more than 20 µg/ml. Information obtained from this study can be used for the identification and standardization of N. nucifera plumule and also toward monograph development on this plant.