The usage of next generation sequencing in combination with targeted gene panels has enforced a better understanding of tumor compositions. The identification of key genomic bi-omarkers underlying a disease are crucial for diagnosis, prognosis, treatment and therapeutic re-sponses. The Oncomine™ Comprehensive Assay v3 (OCAv3) covers 161 cancer‐associated genes and is routinely employed to support clinical decision making for a therapeutic course. An improved version, Oncomine™ Comprehensive Assay Plus (OCA‐Plus), has been recently developed, covering 501 genes (144 overlapping with OCAv3) in addition to microsatellite instability (MSI) and tumor mutational burden (TMB) assays in one workflow. The validation of MSI and TMB was not addressed in the present study. However, the implementation of new assays must be validated and confirmed across multiple samples before it can be introduced into a clinical setting. Here, we report the comparison of DNA sequencing results from 50 ovarian cancer formalin‐fixed, paraffin‐embed-ded samples subjected to OCAv3 and OCA‐Plus. A validation assessment of gene mutations identified using OCA‐Plus was performed on the 144 overlapping genes and 313,769 intersecting nucle-otide positions of the OCAv3 and the OCA‐Plus. Our results showed a 91% concordance within variants classified as likely‐pathogenic or pathogenic. Moreover, results showed that a region of PTEN is poorly covered by the OCA‐Plus assay, hence, we implemented rescue filters for those variants. In conclusion, the OCA‐Plus can reflect the mutational profile of genomic variants com-pared with OCAv3 of 144 overlapping genes, without compromising performance.