Protein microarrays have gained popularity as an attractive tool for various fields, including drug and biomarker development, and diagnostics. Thus, multiplexed binding affinity measurements in microarray format has become crucial. The preparation of microarray-based protein assays relies on precise dispensing of probe solutions to achieve efficient immobilization onto an active surface. The prohibitively high cost of equipment and the need for trained personnel to operate high complexity robotic spotters for microarray fabrication are significant detriments for researchers, especially for small laboratories with limited resources. Here, we present a low-cost, instrument-free dispensing technique by which users who are familiar with micropipetting can manually create multiplexed protein assays that show improved capture efficiency and noise level in comparison to that of the robotically spotted assays. In this study, we compare the efficiency of manually and robotically dispensed α-Lactalbumin probe spots by analyzing the binding kinetics obtained from the interaction with anti-α-Lactalbumin antibodies, using the interferometric reflectance imaging sensor platform. We show that the protein arrays prepared by micropipette manual spotting meet and exceed the performance of those prepared by state-of-the-art robotic spotters. These instrument-free protein assays have higher binding signal (~4-fold improvement) and a ~3-fold better signal-to-noise ratio (SNR) in binding curves, when compared to the data acquired by averaging of 75 robotic spots corresponding to the same effective sensor surface area. We demonstrate the potential of determining antigen-antibody binding coefficients in 24-multiplexed chip format with less than 5% measurement error.