In conventional positron emission tomography (PET), only one radiotracer can be imaged at a time, because all PET isotopes produce the same two 511 keV annihilation photons. Here we describe an image reconstruction method ( multiplexed PET imaging) for the simultaneous in vivo imaging of two PET tracers and thereby the independent quantification of two molecular signals. It is based on the detection of 511 keV annihilation photons and prompt γ-ray emissions within the scanner's energy window. We used multiplexed PET in a large number of studies, such as tracking in mice with subcutaneous tumors the biodistributions of intravenously injected 124I-trametinib and FDG and others, as recently published in Nature Biomed. Engineering. Multiplexed PET provides more information depth, gives new uses to prompt γ-ray-emitting isotopes, reduces radiation burden by omitting the need for an additional computed-tomography scan, and can be implemented on preclinical and clinical systems without any modifications in hardware or image acquisition software.