An in-silico WES approach using the Galaxy platform was adopted in the current study to predict the genetic basis of Premature Ovarian Failure (POF), where three affected patients in a Saudi Arabian family of seven, found associated with X-linked recessive mutations. The current analysis discovered 518,054 variants using FreeBayes variant caller that had 1,461,864 effects on variable sites in the genome revealed by SnpEff software. The causal genetic mutations were filtered and annotated with the ClinVar database using the GEMINI tool. This tool retained 369 pathogenic mutations harboring 130 genes. Among the total, 268 variants positioned on 69 genes are shared with three affected individuals, 61 variants on 23 genes are shared by any two of the affected individuals, and 40 of the variants on 38 genes are present in any one of the affected sample. Two mutations in one of the already POF-associated, POF1B gene were also observed e.g. (i) g.84563135T>A; p.M349L and (ii) g.84563194C>T; p.R329Q in the two affected individuals i.e. IV-I-C & IV-6 in the current data. This gene consists of 17 exons that span the region of >100 kb. The putative function of this gene in regulating the actin cytoskeleton due to homology with myosin tail and maintains a number of oocytes during fetal ovary development. In a nutshell, this Galaxy pipeline facilitates all-in-one to pinpoint not only the known pathogenic gene mutations for this disorder but few other novel genetic variants as well, whose gene-disease association may be validated by further experimental studies.