Male birds of egg-laying hen strains have no commercial value and are culled immediately after hatching, raising concerns for animal welfare. Existing experimental methods for in ovo sexing require taking samples and are applicable after embryos' sexual differentiation. We demonstrate that Raman spectroscopy enables contactless in ovo sex determination of the domestic chicken (Gallus gallus f. dom.) already at day 3.5 of egg incubation. A sexing accuracy of 90% was obtained by analyzing the spectra of blood circulating in the extraembryonic vessels. The measurement is damage-free and barely affects the hatching rate. Sex recognition is achieved before the onset of sensitivity. Therefore, Raman spectroscopy provides an alternative to the culling of 1-day-old male chicks in laying hen production.