A robust object segmentation method for analysis of biological activity receives an input image and performs segmentation confidence mapping using the input image to generate segmentation confidence map output. A thresholding is performed using the object segmentation confidence map to generate a high confidence object mask output. An object segmentation confidence mapping method for analysis of biological activity receives an input image and performs segmentation decision to create segmentation decision result. A difference operation is performed to generate the segmentation decision result. A confidence mapping is performed using the difference result to generate segmentation confidence. An object level robust analysis method for biological activity receives an input image and performs object segmentation using the input image to create object segmentation result. A robust object feature measurement is performed to generate robust object feature result. An FOV level robust analysis method for biological activity receives a plurality of object feature results and performs robust FOV summary feature extraction to create robust FOV summary features. A FOV regulated feature extraction is performed to generate FOV regulated features. A FOV regulated feature extraction method for biological activity receives a plurality of object feature results and performs control object selection using the plurality of object feature results to generate control objects output. A FOV regulated feature extraction is performed to generate FOV regulation features output. An object feature FOV regulation is performed using the plurality of object feature results and the FOV regulation features to generate FOV regulated object features output. A sample level robust analysis method for biological activity receives a plurality of FOV feature results and performs robust sample summary feature extraction to create robust sample summary features. A sample regulated feature extraction is performed to generate sample regulated features. An assay level robust analysis method for biological activity receives a plurality of sample feature results and performs robust assay summary feature extraction to create robust assay summary features. An assay regulated feature extraction is performed to generate assay regulated features.