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Screening biological readouts in cell culture are increasing in frequency and throughput. In such assays, cell types may be rare and reagents or compounds may be expensive often resulting in a reduced number of conditions and/or replicates. “Tubeless” microfluidics offers a method to reduce this burden, as has been previously shown. In addition the In-Cell Western (ICW) has recently been adapted to microfluidic cultures allowing high throughput analysis of immunocytochemistry in microfluidic channels. Combining automated liquid handling in tubeless microfluidics with the ICW provides rapid and quantitative high throughput cell-based screens. Here, we validate this platform using three parameters: operational robustness (pipetting reliability), cell seeding consistency, and cell staining consistency (both nuclear and antibody). Integration of liquid handling with microfluidics was found to be more than 97% operationally robust. Cell seeding consistency between each microchannel and within each microchannel was found to be within a standard deviation of less than 5% and 6%, respectively. Finally, through optimization of liquid handling steps, uniformity among all the channels was found for both nuclear and antibody staining. These results lay the foundation to perform most standard ICW assays using automated tubeless microfluidics. [Copyright &y& Elsevier]