Molecular profiling is a powerful strategy for dissecting molecular aspects of cell functioning. The precision of molecular profiling is highly dependent on purity and homogeneity of cell samples. Cell technologies need cell populations enriched with target cells. Numerous approaches have been proposed to isolate and collect homogenous populations of cells and even single cells for downstream molecular profiling and recultivation. Unlike other methods for isolation of live cells, laser microdissection allows for collecting single cells and cell colonies from adherent cell cultures without detaching cells from the substrate. This is advantageous for subsequent downstream omics analyses or recultivation because of preserving cells in their intact state. Here, we present a protocol for live cell laser microdissection with gravity transfer. This approach allows for isolating fragments of cell monolayers and single cells for downstream molecular profiling and recultivation.