Loss of Tsc1 in the kidneys gives rise to rapid polycystogenesis and kidney failure due to the hyperactivation of mTOR complex I (mTORC1). One of the major downstream targets of mTORC1 is S6K1, which is responsible for driving many of the effector functions of mTOR. Strikingly, the loss of S6k1 in the context of hyperactivated mTOR is sufficient to reverse the generation of polycystic kidneys. In an effort to uncover novel direct S6k1 substrates involved in the generation of cystic kidneys, we generated Tsc1-null and Tsc1/S6k1-double null murine inner medullary collecting duct (iMCD3) cell lines using CRISPR/Cas9. Using these two cell lines, we conducted a phosphoproteome screen to search for novel candidates. Using a combination of antibody-motif enrichment and metal affinity enrichment, we provide a comprehensive phosphoproteome profile between these two genotypes.