High throughput studies have revealed the pervasive nature of transcription in eukaryotic genomes, resulting in the identification of a multitude of non-coding RNAs (ncRNAs). In yeast, a substantial number of ncRNAs overlap protein-coding genes in antisense direction. While numerous reports on antisense-dependent gene regulation have been published, most studies focus on a few selected cases or rely on correlations derived from RNA-based high throughput experiments. Therefore, our understanding of which antisense RNAs exert a biological function and whether functional antisense RNAs share certain features remains incomplete. To address these shortcomings, we developed a genetic strategy to specifically abrogate antisense transcription in selected genomic loci and to directly measure the resulting impact on sense protein levels using GFP as a reporter. To gain a systematic understanding, we applied this technique to 162 yeast genes known to be overlapped by antisense RNAs. Subsequently, we used quantitative high throughput microscopy to score the effects of each tested antisense transcript on sense protein abundance under several growth conditions, thus discriminating between functional and non-functional antisense RNAs. We found that only a minority of the genes (20%) were affected by antisense transcription and that in those cases regulation was generally weak and led to a reduction in protein amounts. Interestingly, antisense regulated genes also showed reduced protein expression noise. When investigating whether certain features distinguish functional from non-functional antisense RNAs we found that the functional antisense transcripts were more likely to extend into the transcript start site of the sense. The effect of antisense increased at high antisense RNA levels but was reduced at high sense RNA levels. While we could not identify any specific sequence motifs, we found that antisense regulated genes had increased H3K4 di- and trimethylation levels. Our data shed light on the general principles that underlie antisense-mediated gene expression regulation and will provide a rich resource for detailed mechanistic studies.