Experimental research on microfluidic devices requires adequate control over surface parameters like wettability. Plasma has already been proven to be a promising tool for the control and alteration of the wettability of solid surfaces, yet its propagation in microfluidic devices and treatment stability remains challenging. Our idea is to produce and propagate an atmospheric pressure helium plasma directly into closed micrometer-size glass channels for in situ wettability treatment. This approach enables better control over the treatment parameters compared to conventional treatments in low-pressure chamber-type plasma reactors. With a homemade kHz dielectric barrier discharge-like setup, we successfully propagated plasma through a \(4\,\hbox {cm}\) long rectangular microchannel of uniform depth (\(100\,\upmu \hbox {m}\)) and variable width (250–500 \(\,\upmu \hbox {m}\)). Results obtained by in situ contact angle measurement on images indicate uniform wettability treatment with increased hydrophilic properties after only 1 min of treatment. The wettability achieved on a glass with our setup offers stability for up to 70 days depending on the plasma treatment and storage parameters. Contact angle results are further supported with X-ray photoelectron spectroscopy (XPS) surface analysis which revealed that the two effective mechanisms for wettability alteration are cleaning and surface functionalization.