Irinotecan (IRI) is utilised as a first line anticancer medication in the cure of cancer having extraordinary ability to block DNA-topoisomerase-I. It is used as a monotherapy and adjunct therapy in the treatment of metastatic colorectal cancer and other cancers, and it differs chemically and pharmacologically from other anticancer medications. The proposed review is divided into two main sections i.e. a) Different analytical methods for estimating irinotecan in pharmaceutical formulations, b) Diverse analytical methods for detecting irinotecan in biological matrices. This work also considers the development of numerous analytical methods based on various parameters, as well as the validation of the methods used. Estimated validation characteristics such as Linearity, Limit of Detection (LOD), and Limit of Quantitation (LOQ) are considered for each. Applying bioanalytical methods, the wavelength of detection, mobile phase, columns, flow rate, retention duration, and sample preparation processes are all evaluated as essential quality variables for estimating Irinotecan.