In this study, the influence of the presence of excipients in sample preparation and clean-up steps required prior to drug analysis in milk-based media which simulate the in vivo properties of the fed state stomach was investigated. 15 excipients, normally present in solid dosage forms of five APIs tested (atenolol, paracetamol, furosemide, nifedipine and propafenone hydrochloride) were mixed (one at a time) with the active pharmaceutical ingredient of interest either via vortexing, co-grinding or shaking of the physical mixture and dissolved in Fed State Simulated Gastric Fluid (FeSSGF). The objective of the study was the assessment of the extraction efficiency of three protein precipitation protocols (using MeOH, ΑCN and 10% w/v TCA), typically used in drug analysis, in milk-based biorelevant media in the presence of the excipients. The mixing technique, fat content of the medium and excipient and solvent effects were investigated. The efficiency of three different protein precipitation reagents in drug extraction when dissolved as API:excipient mixtures in the fed-state medium was compared against the equivalent drug amount recovered in the absence of the excipient in FeSSGF. Most excipients had a significant negative effect (p < 0.05) on drug recovery in the milk-based medium as indicated by the multiple linear regression (MLR) analysis performed. For magnesium stearate and HPMC, the % recovery values were the lowest in four out of the five drugs studied, with a range of 10-100% depending on the API, mixing technique and protein precipitation protocol selected. The negative excipient-dependent effect was more profound in nifedipine and propafenone hydrochloride, the most lipophilic compounds of the study. Acetonitrile was the most effective extraction reagent for most drugs in the presence of excipients, followed by methanol and 10% w/v trichloroacetic acid. Data analysis also revealed a dependence of the extraction method efficiency on the medium lipid content. Application of the above extraction protocols in commercially available formulations highlighted the need for assessment of the effect of excipients in extraction efficiency, before transferring the method directly to dissolution studies of formulations in milk-based fed gastric media. In conclusion, the presence of excipients and the selection of protein precipitation protocol are parameters which can affect significantly the efficiency of protein precipitation when FeSSGF is used as dissolution medium and need to be taken into consideration when developing a quantitative method based on the above sample clean-up technique.