Amastigotes are the replicative intracellular life cycle stage of the protozoan parasite Trypanosoma cruzi, the causative agent of Chagas disease. They have the ability to proliferate in any nucleated mammalian cell. A greater understanding of amastigote biology would greatly aid drug development since this life cycle stage is the principal target of chemotherapy. Reports have linked recrudescence following drug treatment with the possible existence of a quiescent amastigote phenotype. However, the lack of a rapid and straightforward method for isolating amastigotes from infected host cells has limited research progress in this area. This is particularly the case with omics technology, where the current complex fractionation and purification procedures can act to perturb RNA and protein expression. Here, we outline a methodology that largely overcomes these problems. Our protocol exploits MP Bio-Lysing Matrix M tubes to promote the differential lysis of host cells and the release of intact amastigotes in a 1-min time frame. Immediate treatment of the lysate with CellCover reagent then maintains RNA and protein in their native states. Amastigotes stabilized in this way can undergo further manipulations, such as cell sorting, without modification of their proteome or transcriptome profiles. This methodology, which is flexible and widely applicable, should greatly benefit research into the intracellular life cycle of T. cruzi.