Background: The success of drugs targeting the MAPK or PI3K pathways in cancer has been slowed by insufficient pathway suppression or onset of resistance through rebound or cross-pathway activation. Isoform-specific measurement of key signaling molecules and a pathway convergence marker ribosomal protein S6 (RPS6), may provide valuable pharmacodynamic (PD) evidence to assess drug effectiveness. Methods: We developed Luminex multiplex assays to measure total and phosphorylated forms of RPS6, ERK1, ERK2, MEK1, MEK2, AKT1, AKT2, and AKT3 from a single tumor biopsy. Fit-for-purpose validation was performed with three drugs currently in clinical trials. A SW620 (KRAS G12V) model was tested with selumetinib (NSC741078, 20 mg/kg), MK2206 (NSC756656, 24 mg/kg), and two PTEN-null models (PC3 & HCC70) with AZD8186 (NSC777572, 25 & 50 mg/kg). Tumors were collected at multiple timepoints after single and repeated dosing, processed by a validated method (PMID 27001313) and data analyzed relative to vehicle control (n = 4-5/grp). Results: The multiplexed assays demonstrated satisfactory analytical performance. Treatment of the SW620 model with a single dose of selumetinib suppressed pERK1/2 ( > 90%), MEK1/2 (50%) and pRPS6 (40-60%) up to 4 h (last time point tested) which was reversed by 24 h. AKT inhibitor, MK2206, suppressed pAKT1/2 (75%) and pAKT3 (50%) 2 h post-dose with reversal at 24 h but did not significantly suppress pRPS6, implying limited downstream modulation. Dosing selumetinib for 21 days had no added effect on pERK isoforms. In both PTEN-null models, AZD8186 suppressed all pAKT isoforms by 40-75% up to 4 h post-dose. The pAKT remained suppressed at 7 h in HCC70 but both pAKT and pRPS6 rebounded by 7 h in PC3. The basal pERK1/2 levels were low and unaffected by AZD8186 in PC3 and HCC70. Conclusions: We established the fitness of PD assays to provide critical evidence regarding the duration of on-target effects, timeline of biomarker reversal and effectiveness of pathway suppression for three distinct classes of drugs. The PD assays are ready for an ongoing clinical trial of AZD8186 and to support clinical development of FGFR inhibitors. Funded by NCI Contract HHSN261200800001E.