One of the most significant challenges impeding cancer treatment effectiveness is drug resistance. Combining evolutionary understanding with drug resistance can pave the way for the identification of second-line drug options that can overcome drug resistance. Although capecitabine and irinotecan are commonly used therapeutic agents in the treatment of CRC patients, resistance to these agents is common. The underlying clonal dynamics of resistance to these agents using high-resolution barcode technology and identification of effective second-line drugs in this context remain unclear. Caco-2 and HT-29 cell lines were barcoded, and then capecitabine and irinotecan resistant derivatives of these cell lines were established. The frequencies of barcodes from resistant cell lines and harvested medium, longitudinally, were determined. Collateral drug sensitivity testing was carried out on resistant Caco-2 and HT-29 cell lines using single agents or drug combinations. The SyngeryFinder tool was used to analyse drug combination testing. In Caco-2 and HT-29 cell lines, barcode frequency measurements revealed clonal dynamics of capecitabine and irinotecan formed by both pre-existing and barcodes, indicating the presence of polyclonal drug resistance. The temporal dynamics of clonal evolution in Caco-2 and HT-29 cell lines were demonstrated by longitudinal analysis of pre-existing and barcodes from harvested medium. In Caco-2 and HT-29 cell lines, collateral drug sensitivity revealed a number of drugs that were effective alone and in combination. The use of barcoding technology reveals the clonal dynamics of chemotherapy-induced drug resistance not only from harvested cell populations, but also from longitudinal sampling throughout the course of clonal evolution. Second-line drugs that sensitize drug-resistant CRC cell lines are identified through collateral drug testing.