Methods and compositions for detecting cross-contamination between samples contacted with different arrays of a multi-array substrate are provided. The methods involve contacting sample to arrays of a multi-array substrate that contains cross-contamination probes in each of its arrays, and evaluating the resultant sample contacted arrays for cross-contamination between the samples. In many embodiments, the arrays of the multi-array substrate contain a set of cross-contamination probes for a corresponding set of cross-contamination targets in the sample(s). Kits and systems are provided for performing the invention. The subject methods may be used in a variety of different applications, such as gene expression analysis, DNA sequencing, mutation detection, as well as other genomics and proteomics applications.