Plant steroids constitute a diverse group of natural products. Biosynthetically, they are derived from
S
‐squalene‐2,3‐epoxide via acetate‐mevalonate pathway. Among the plant steroids, phytosterols are ubiquitous in the plant kingdom and some of them are reported to possess hypocholesterolemic activity. Withanolides are a large group of steroidal lactones with various biological activities. Brassinosteroids are a small group of plant steroids classified as plant growth hormones. Phytoecdysteroids are polyhydroxylated plant steroids, many of which are known to exhibit anabolic effects with no undesirable side effects. Steroidal alkaloids are nitrogen‐containing plant steroids with an array of biological activities. It is noteworthy that trace amounts of cholesterol and mammalian steroidal hormones including progesterone have been detected in some plants. Analyses of plant steroids mainly utilize chromatographic methods such as
thin‐layer chromatography
(
TLC
),
high‐performance liquid chromatography
(
HPLC
),
ultra‐high‐performance liquid chromatography
(
UHPLC
), and
gas‐liquid chromatography
(
GLC
). In general,
photodiode array
(
PDA
),
evaporative light scattering
(
ELS
), and
fluorescence
(
FL
) detectors and
mass spectrometry
(
MS
) are commonly used for detection, quantification, and identification of plant steroids. UHPLC hyphenated with
tandem mass spectrometry
(
MS/MS
) has recently become the state‐of‐the‐art method of choice for the analysis of plant steroids, especially for the ones that are present in minor quantities such as brassinosteroids and phytoecdysteroids. Some less common techniques employed for the analyses of plant steroids are immunoassays and biological assays.