Multiple elements in human sperm have been demonstrated to play significant roles in the reproductive process, but their simultaneous detection in single cells remains challenging. We propose a novel analytical procedure using single-cell inductively coupled plasma-time of flight-mass spectrometry (scICP-TOF-MS) to simultaneously quantify multiple elements of individual sperm cells. A promising label-free cell identification strategy based on the endogenous element was developed to obtain valid data. The element contents exhibited varied degrees of heterogeneity in single cells. Machine learning-based analysis of the multi-dimension dataset indicated different distribution patterns and physiological roles among the simultaneously detected elements.