The bulbs of the Easter lily ( Lilium longiflorum Thunb.) are regularly consumed in Asia as both food and medicine, and the beautiful white flowers are appreciated worldwide as an attractive ornamental. The Easter lily is a rich source of steroidal glycosides, a group of compounds that may be responsible for some of the traditional medicinal uses of lilies. Since the appearance of recent reports on the role steroidal glycosides in animal and human health, there is increasing interest in the concentration of these natural products in plant-derived foods. A LC-MS/MS method performed in multiple reaction monitoring (MRM) mode was used for the quantitative analysis of two steroidal glycoalkaloids and three furostanol saponins, in the different organs of L. longiflorum. The highest concentrations of the total five steroidal glycosides were 12.02 ± 0.36, 10.09 ± 0.23, and 9.36 ± 0.27 mg/g dry weight in flower buds, lower stems, and leaves, respectively. The highest concentrations of the two steroidal glycoalkaloids were 8.49 ± 0.3, 6.91 ± 0.22, and 5.83 ± 0.15 mg/g dry weight in flower buds, leaves, and bulbs, respectively. In contrast, the highest concentrations of the three furostanol saponins were 4.87 ± 0.13, 4.37 ± 0.07, and 3.53 ± 0.06 mg/g dry weight in lower stems, fleshy roots, and flower buds, respectively. The steroidal glycoalkaloids were detected in higher concentrations as compared to the furostanol saponins in all of the plant organs except the roots. The ratio of the steroidal glycoalkaloids to furostanol saponins was higher in the plant organs exposed to light and decreased in proportion from the aboveground organs to the underground organs. Additionally, histological staining of bulb scales revealed differential furostanol accumulation in the basal plate, bulb scale epidermal cells, and vascular bundles, with little or no staining in the mesophyll of the bulb scale. An understanding of the distribution of steroidal glycosides in the different organs of L. longiflorum is the first step in developing insight into the role these compounds play in plant biology and chemical ecology and aids in the development of extraction and purification methodologies for food, health, and industrial applications. In the present study, (22R,25R)-spirosol-5-en-3β-yl O-α-l-rhamnopyranosyl-(1→2)-β-d-glucopyranosyl-(1→4)-β-d-glucopyranoside, (22R,25R)-spirosol-5-en-3β-yl O-α-l-rhamnopyranosyl-(1→2)-[6-O-acetyl-β-d-glucopyranosyl-(1→4)]-β-d-glucopyranoside, (25R)-26-O-(β-d-glucopyranosyl)furost-5-ene-3β,22α,26-triol 3-O-α-l-rhamnopyranosyl-(1→2)-β-d-glucopyranosyl-(1→4)-β-d-glucopyranoside, (25R)-26-O-(β-d-glucopyranosyl)furost-5-ene-3β,22α,26-triol 3-O-α-l-rhamnopyranosyl-(1→2)-α-l-arabinopyranosyl-(1→3)-β-d-glucopyranoside, and (25R)-26-O-(β-d-glucopyranosyl)furost-5-ene-3β,22α,26-triol 3-O-α-l-rhamnopyranosyl-(1→2)-α-l-xylopyranosyl-(1→3)-β-d-glucopyranoside were quantified in the different organs of L. longiflorum for the first time.