{Reference Type}: Journal Article {Title}: Analytical Strategies to Investigate Molecular Signaling, Proteomics, Extraction and Quantification of Withanolides - A Comprehensive Review. {Author}: Gopalaiah SB;Jayaseelan K; {Journal}: Crit Rev Anal Chem {Volume}: 0 {Issue}: 0 {Year}: 2024 Feb 1 {Factor}: 5.686 {DOI}: 10.1080/10408347.2024.2307887 {Abstract}: Withanolides are the class of steroidal molecules getting greater emphasis in recent years. Quality control throughout the manufacturing and storage period is often one of the key problems that have restricted their broad use in India's indigenous and Ayurvedic medical systems for thousands of years. Because of their diverse clinical potential, withanolides have received a great deal of scientific attention. Analytical techniques are being devised for the automated isolation, identification, and estimation of every single protein within the cell as well as in herbal extracts of withanolides, due to which now researchers are interested in determining the effects of metabolism as well as various stimuli on protein expression, which made the study easier. This study discusses the potential use of hyphenated analytical methods that are reliable in understanding the molecular signaling features, proteome evaluation and characterization of withanolides, in addition to examining existing methodological limitations. The choice of analytical techniques for the withanolides analysis, however, relies on the nature of the sample matrix, the aim of the analysis, and the sensitivity of the technique.