ABSTRACT Mosaic loss of the Y chromosome (mLOY) is the most common somatic event in men, strongly associated with aging and various health conditions. Current methods for detecting mLOY primarily rely on DNA genotyping arrays. Here, we present MosCoverY, a novel method for estimating mLOY from NGS sequencing data that can be applied to both exome and genome sequencing. MosCoverY addresses the challenges posed by the structure of the Y chromosome by focusing on single-copy genes and normalizing their coverage against autosomal exons matched by length and GC content. We validated MosCoverY using data from 212,062 male participants in the UK Biobank, comparing its results to those obtained using genotyping- or whole genome sequencing-based methods. MosCoverY identified mLOY in 5.6% of men, demonstrating performance that was comparable to the other methods. MosCoverY also replicated known associations between mLOY, age, smoking, all-cause mortality, and germline genetic loci, showing the strongest associations in many cases. MosCoverY offers a valuable tool for detecting mLOY from exome data in population-scale studies.