ABHD2 is a serine hydrolase that belongs to the subgroup of the α,β-hydrolase fold containing proteins, which is involved in virus propagation, immune response and fertilization. Chemical tools to selectively modulate the activity of ABHD2 in an acute setting are highly desired to investigate its biological role, but are currently lacking. Here, we report a library versus library screening using activity-based protein profiling (ABPP) to evaluate in parallel the selectivity and activity of a focused lipase inhibitor library against ABHD2 and a panel of closely related ABHD proteins. This screen resulted in the rapid identification of novel inhibitors for ABHD2. The selectivity of the inhibitor was further investigated in native mouse testis proteome by competitive ABPP, revealing a highly restricted off-target profile. Progesterone-induced acrosome reaction was reduced in a dose-dependent manner by the newly identified inhibitor, which provides further support for the key-role of ABHD2 in P4-stimulated acrosome reaction. On this basis, the ABHD2 inhibitor is an excellent starting point for further optimization of ABHD2 inhibitors that can modulate sperm fertility and may lead to novel contraceptives.