In animal research, a strong bias towards using male animals is apparent in most disciplines. By using animals of only one sex, researchers risk missing important sex differences and using animals for inconclusive findings. In response to such overt sex bias, major funders now request the inclusion of sex as a biological variable in all animal experiments. However, this poses challenges to the 3Rs: experiments may require up to twice as many animals and researchers face difficult logistical decisions with important implications for both animal welfare and scientific validity. When including both sexes, male and female animals may be housed either with or without social cues from conspecifics of the other sex. Although this may profoundly affect the animals' physiology and behaviour, with implications for both scientific validity and animal welfare, it is frequently ignored and rarely reported in publications. Currently, there is no consensus on best practices and guidelines are missing. To address these challenges and inform best practice in terms of scientific validity, animal welfare, and reducing animal use, we will systematically study in male and female mice how mixed-sex vs same-sex housing affects sex-dependent biological functions, measures of animal welfare, and variation in experimental results under both open-top cage and IVC conditions, and develop tools for dissemination of best practice. As the inclusion of sex as a biological variable concerns all areas of animal research and mice account for over 70% of all animals used in research, the impact of our findings on the 3Rs may be huge.