Here we present a water-in-air droplet platform for micro-compartmentalization for single molecule guided synthesis and analysis consisting of a flow-system hosting dense arrays of aqueous microdroplets on a glass surface surrounded by air. The droplets are formed in a few seconds by passing a waterfront over the array of hydrophilic spots surrounded by a hydrophobic coating, thus forming a micro-droplet array (MDA). The droplet volumes are tunable from approximately 50 femtoliter to 20 picoliter by adjusting the size of the hydrophilic spots. MDAs consisting of femtoliter volume droplets were stable for more than 24 hours in air at 37 °C in a reversibly sealed flow-system, thus allowing us to perform assays that require long incubations in the droplets. Using differently fluorescing liquids, it was further shown that droplets can be reformed on the same MDA several times by passing a new liquid plug over the surface, and that fluorescence from one reaction can be washed away with little to no carry-over, hence allowing for multistep reactions to be carried out on the system. The MDA created by an air/water interface supported digital immunoassays as was demonstrated by measuring the Aβ42 peptide in cerebrospinal fluid of Alzheimers patients and control patients. To demonstrate a two step droplet assay, first, histidine tagged peptides were expressed in the droplets and bound to the droplet-enclosed surface. Subsequently, the his-tagged peptides were detected using enzyme-conjugated antibodies in a second droplet generation step. As such, the chip demonstrates features necessary for library preparations for high throughput screening applications.