Human skin is a complex material to test and model as its physical and geometric properties depend on a host of parameters: thickness, location, age, ethnicity, hydration, etc. Destructive testing of skin samples only gives a partial picture, because harvesting skin dehydrates the sample and releases its residual stress, which is likely to alter its behaviour significantly. Knowledge of the in vivo stress in skin will aid in preoperative surgery planning, e.g., by providing safe limits of skin stress or by accurately estimating the area of skin for defect repairs. Here, we develop and validate a method to quantify the in vivo stress in skin using acoustic measurements. We find that it is possible to determine the difference in stress along the in-plane directions on a patient-specific basis, using a simple in vivo measurement technique. We model the skin as an incompressible, anisotropic hyperelastic material with one family of fibres, with the principal pre-stress aligned along the fibre direction [1]. A detailed analysis reveals that the in-plane stress difference is related to the surface wave speeds, via a simple formula that does not assume material properties, with a known error of less than 9%. We validate the formula with finite element simulations using Abaqus, where symmetry conditions are employed to reduce computational cost. The in vivo stress is replicated by applying a pre-stretch and the wave is induced by an instantaneous impulse. We then measure the wave speeds parallel and perpendicular to the fibres at various levels of pre-stress. We find that the error between the actual stress difference and the stress difference calculated with the method is less than 5%, for simulations using the Holzapfel-Gasser-Ogden model [2], which is within the range determined by the method. The proposed method is universal and can be used to determine the in vivo stress difference, independent of material properties or energy density function. In vivo stress is difficult to estimate in general, however, the method proposed here will enable on-demand patient-specific measurement of the in vivo stress in skin in a simple, non-invasive manner using easily accessible parameters. This method could replace existing qualitative techniques with more accurate quantitative measurements, improving preoperative reconstructive surgery planning and ultimately improving surgical outcomes.