Autophagy is a cellular mechanism that degrades damaged organelles and misfolded proteins to maintain cellular homeostasis. Autophagy in cancers is drawing increasing attentions due to its multifaceted roles in cancer development, progression, and treatment. There are several key autophagy effectors that are being extensively studied to understand the role of autophagy in cancer as well as their potential value as predictive and/or prognostic biomarkers and therapeutic target. These include ATG4A, ATG4B, Beclin-I, p62, LC3A, LC3B, LC3C, and LAMP. While having its own sophisticated pathway, autophagy has been reported to associate with multiple oncogenic pathways such as NF-kB, mTOR, and PI3K signaling. This chapter aims to provide a detailed protocol for researchers to investigate the role of autophagy using in vitro cell line as model. Here, we demonstrate several techniques including Western blot (WB), immunofluorescence (IF), and small-interfering RNA (siRNA) knockdown using colorectal cancer cell lines as samples. This chapter provides information to researchers especially those in their earlyand mid-career to plan and design their experiments to study the autophagy events in their area of interests. 1.In a 24-well plate, the cells were seeded at 50% confluency and left to incubate overnight.2. The media were then changed accordingly, with normal media, serum-free media, and media with the inhibitor. 3.After 24-hour incubation, the cells were collected and rinsed with PBS. 4.The cells were then lysed with lysis cocktail consisting of 1X RIPA lysis buffer and 1X protease inhibitor cocktail. 5.The lysates were vortexed briefly every 5 minutes for 30 minutes and kept in ice in between mixing. 6.The lysates were then centrifuged at 16000×g for 15 minutes at 4°C, and the supernatants were kept. 7.The lysates were then quantified by using BCA protein assay and immediately diluted with deionized water to obtain 13 μL of 20 μg lysate and then added to 5 μL of 4X SDS loading buffer and 2 μL of 2-mercaptoethanol.8. The samples were boiled at 90°C for 10 minutes, and 10 μL of the samples were loaded into the well of a gradient gel.9. The lysates were separated by SDS electrophoresis by using Tris/Glycine/SDS running buffer at 150 V for approximately 45 minutes and transferred to a PVDF membrane by using FlashBlot transfer buffer at 55 V for 1 hour. 10.The membrane was then blocked by using 5% Blotting Grade Blocker in TBST for 1 hour to prevent non-specific binding of antibody. 11.The membrane was then incubated with primary antibody diluted with the Blocker in TBST at 1:2000 dilution. 12.The incubation was done overnight at 4°C or 1 hour at room temperature.13. Any unbound antibodies were then removed by washing with TBST for 10 minutes once and 5 minutes for another 2 times. 14.Secondary antibodies conjugated with horseradish peroxidase (HRP) were then prepared by diluting the antibody in the Blocker in TBST at 1:3000 dilution. 15.The membrane was then incubated in the secondary antibody:a. Anti-rabbit secondary antib...