Epithelial-mesenchymal transition (EMT), a crucial mechanism in carcinoma progression, describes the process whereby epithelial cells lose their apico-basal polarity and junctional complexes and acquire a mesenchymal-like morphology. Several markers are considered to be authentic indicators of an epithelial or mesenchymal status; however, there is currently no comprehensive or systematic method with which to determine their functional relevance. Previously, we identified a 33-gene EMT signature comprising 25 epithelial and 6 mesenchymal genes that best describe this concept of the EMT spectrum. Here, we designed small-scale siRNA screens targeting these six mesenchymal signature genes (CD99L2, EMP3, ITGA5, SYDE1, VIM, ZEB1) to explore their functional relevance and their roles during EMT reversal by nintedanib (BIBF1120) in a mesenchymal-like SKOV3 ovarian cancer cell line. We found that neither cell proliferation nor cytotoxicity was affected by silencing any of these genes. SKOV3 cells expressing siRNA against mesenchymal genes (ZEB1, EMP3, CD99L2, ITGA5, and SYDE1) showed enhanced colony compaction (reduced inter-nuclear distance). Inductions of E-cadherin expression were only observed in SYDE1- and ZEB1-silenced SKOV3 cells. In addition, only SYDE1-silenced SKOV3 cells showed increased anoikis. Finally, we identified that SYDE1 and ZEB1 were down-regulated in nintedanib-treated SKOV3 cells and SYDE1- and ZEB1-silenced SKOV3 cells showed enhanced nintedanib-induced up-regulation of E-cadherin. Nintedanib-treated SKOV3 cells also showed colony compaction and decreases in EMT scores both in vitro and in vivo. We conclude that SYDE1 and ZEB1 are functionally relevant in EMT reversal. This study thus provides a proof-of-concept for the use of in vitro siRNA screening to explore the EMT-related functions of selected genes and their potential relevance in the discovery of EMT reversing drugs.