Label-free biosensors enable the monitoring of biomolecular interactions in real time, which is key to the analysis of the binding characteristics of biomolecules. While refractometric optical biosensors such as surface plasmon resonance (SPR) are sensitive and well-established, they are susceptible to any change of the refractive index in the sensing volume caused by minute variations in composition of the sample buffer, temperature drifts, and most importantly nonspecific binding to the sensor surface in complex fluids such as blood. The limitations arise because refractometric sensors measure the refractive index of the entire sensing volume. Conversely, diffractometric biosensors–for example, focal molography–only detect the diffracted light from a coherent assembly of analyte molecules. Thus any refractive index distribution that is noncoherent with respect to this molecular assembly does not add to the coherent signal. This makes diffractometric biosensors inherently robust and enables sensitive measurements without reference channels or temperature stabilization. The coherent assembly is generated by selective binding of the analyte molecules to a synthetic binding pattern–the mologram. Focal molography has been introduced theoretically [C. Fattinger, Phys. Rev. X 4, 031024 (2014)] and verified experimentally [V. Gatterdam, A. Frutiger, K.-P. Stengele, D. Heindl, T. Lübbes, J. Vörös, and C. Fattinger, Nat. Nanotechnol. 12, 1089 (2017)] in previous papers. However, further understanding of the underlying physics and a diffraction-limited readout is needed to unveil its full potential. This paper introduces refined theoretical models, which can accurately quantify the amount of biological matter bound to the mologram from the diffracted intensity. In addition, it presents measurements of diffraction-limited molographic foci, i.e., Airy discs. These improvements enable us to demonstrate a resolution in real-time binding experiments comparable to the best SPR sensors without the need for temperature stabilization or drift correction and to detect low-molecular-weight compounds label free in an endpoint format. The presented experiments exemplify the robustness and sensitivity of the diffractometric sensor principle.