A method for determining the mated status of female fruit flies, focusing on determining the presence of spermatozoa in a sample fly's ventral receptacle (VR). The ventral receptacle is the organ where eggs become fertilized. The VR is the first and last organ in a mated female fly to contain spermatozoa. The VR is thus the most accurate organ for determining whether a female fruit fly has recently mated. The method is a squash method. The fly's ventral receptacle is isolated, and then squeezed so as to cause spermatozoa stored in the ventral receptacle to be released into the lumen of the VR. This permits use of a microscope to determine the presence or absence of spermatozoa in the female fruit fly, and thus the mated status of the female fruit fly.