Calabash fruit (Crescentia cujete L.) is one of the herbs cultivated in Indonesia. Calabash fruit has nutrients, including choline, required for neurotransmitter synthesis in neuronal cells. However, the choline levels in fresh fruits decrease during storage. Therefore, there is a need to isolate choline and maintain its level during storage. This study aimed to analyze the effects of extraction, fermentation, and storage processes on the level of choline derived from the calabashh fruit. Calabash fruits were classified into three groups: group I (fresh fruit), group II (extracted), and group III (fermented). All groups were stored in the refrigerator at 4 ℃. Their choline content was measured using liquid chromatography-tandem mass spectrometry on days 0, 5, and 10 after storage. The data were analyzed using the Statistical Package for Social Sciences version 26. The result showed that the calibration curve equation of choline was y = 3.15e + 5x + 2.0e+4, with R2 = 0.998 and a linear regression of concentration range of 0-24 ng/mL. The processing of calabash fruits using extraction or fermentation decreased the choline level (p ≤ 0.05). However, the product produced during extraction and fermentation of the calabash fruit had better stability regarding its choline levels after 10 days of storage compared with that of the fresh calabash fruit (p ≤ 0.05). Therefore, the calabash fruit extraction and fermentation processes can bind choline and maintain its stability after storage. The extraction and fermentation produce products containing pure choline with minimum interfering compounds that affect its stability.